WO2022135753A1 - Methods for prognosis the humoral response of a subject prior to vaccination - Google Patents

Abstract

The present invention represents the first systems biology approach to investigate the volunteers' immune predisposition to respond to a vaccination (MVA-B), assessed by their blood transcriptome profile; specifically, that related to their B cell differentiation stages, and its conditioning by the human microbiota before vaccination. That is, inventors investigated the host gene expression in blood by a microarray approach and the skin and stool microbiota both before vaccination. The objective was to examine their potential involvement in an effective MVA-B neutralizing antibody (Nabs) response during a randomized phase Ib clinical study. This trial immunized 10 HIV seronegative subjects aged from 18 to 45 years by the intramuscular route with MVA-HIV clade B vaccine. Inventors found that gene expression of a group of genes (detected through mRNA nucleic acids in blood) are overexpressed at baseline in subjects with the lowest antibody response compared to the subjects with the highest antibody response, and therefore may be considered as good biomarkers to assess the humoral immune response of a subject to a vaccine. Accordingly, the present invention relates to methods and kits for predicting the humoral response of a subject prior to vaccination. More specifically present invention relates to methods for assessing the humoral response of a subject to a vaccine through detection in a blood sample of specific RNAs.

Description

METHODS FOR PROGNOSIS THE HUMORAL RESPONSE OF A SUBJECT PRIOR TO VACCINATION

FIELD OF THE INVENTION:

The present invention relates to methods and kits for predicting the humoral response of a subject prior to vaccination. More specifically present invention relates to methods for assessing the humoral response of a subject to a vaccine through detection in a blood sample of specific RNAs.

BACKGROUND OF THE INVENTION:

Systems biology has been successfully used to investigate the fundamental innate immune mechanisms orchestrating protective adaptive responses after the perturbation of vaccination against yellow fever (Gaucher et al., 2008; Querec et al., 2009), HIV (Ehrenberg et al., 2019), Ebola (Rechtien et al., 2017), and influenza (Nakaya et al., 2011). An important challenge, however, is to analyze individual baseline human health characteristics to help identify those at higher risk of infection despite vaccination. Until now, only a few studies have looked for candidate traits associated with vaccine responsiveness and partially predicting the humoral response to vaccination against influenza (Furman et al., 2014; Parvandeh et al., 2019; Team and Consortium, 2017; Tsang et al., 2014). No study has examined the interrelations between each individual's immunological state, their microbiota at baseline, and the impact of both on their vaccine-induced immune responses. As the most successful vaccines act through the production of antibodies (Plotkin, 2010), identifying specific individual characteristics of the genome at baseline should enhance our ability for dividing vaccinees into “high responders” or “low responders” in terms of humoral response (Tsang, 2015). Such predictive markers might serve as a potential diagnostic tool that assists vaccine development by taking into account the interindividual heterogeneity of immune responses.

Accordingly, there remains an unmet need in the art for specific and more rapid prognostic test for predicting the humoral response of a subject prior to vaccination, reflecting the immunological status of the patient. SUMMARY OF THE INVENTION:

The present invention relates to an in vitro method for assessing a subject’s humoral immune response to a vaccine, said method, comprising the step of (a) measuring in a sample obtained from said subject prior to the administration of said vaccine the level of one or more gene expression level selected from a group of genes consisting of: IGLV8-61, BLK, EBF1 and IFNal6 gene b) wherein the level of one or more gene expression of step a) is negatively correlated with the humoral immune response of said subject.

Another object of the invention relates to a kit, comprising:

- at least a mean for determining one or more gene expression level selected from a group of gene consisting of: IGLV8-61, BLK, EBF1 and IFNal6 gene and

- instructions for use, for use to assess a subject’s humoral immune response to a vaccine.

DETAILED DESCRIPTION OF THE INVENTION:

Prognostic methods according to the invention

Inventors work represents the first systems biology approach to investigate the volunteers' immune predisposition to respond to MVA-B vaccination, assessed by their blood transcriptome profile; specifically, that related to their B cell differentiation stages, and its conditioning by the human microbiota before vaccination. That is, they investigated the host gene expression in blood by a microarray approach and the skin and stool microbiota by using 16S ribosomal RNA sequencing both before vaccination. The objective was to examine their potential involvement in an effective MVA-B neutralizing antibody (Nabs) response during the CUTHIVAC 03 randomized phase lb clinical study. This trial immunized 10 HIV seronegative subjects aged from 18 to 45 years by the intramuscular route with MVA-HIV clade B vaccine. Inventors analyzed their baseline transcriptomic signature in blood and baseline bacterial abundance and diversity in skin and stool to assess their potential association with the intensity of the Nabs response.

In other words, the inventors found that gene expression of a group of genes of the invention (detected through mRNA nucleic acids in blood) are overexpressed at baseline in subjects with the lowest antibody response compared to the subjects with the highest antibody response, and therefore may be considered as good biomarkers to assess the humoral immune response of a subject to a vaccine (Figure 1C and 2A, 2B, 2C, 2D, 2E).

This minimal marker set identified by the inventors, may be used as prognoses tool alone or in combination with bacterial abundance and diversity scores. These results thus set- up the basis for the development of a rapid functional specific prognostic test for predicting the humoral response of a subject prior to vaccination with an antigen.

Thus, the present invention relates to an in vitro method for assessing a subject’s humoral immune response to a vaccine, said method, comprising the step of (a) measuring in a sample obtained from said subject prior to the administration of said vaccine, the level of one or more gene expression level selected from a group of genes consisting of: IGLV8-61, BLK, EBF1 and IFNal6 gene b) wherein the level of one or more gene expression of step a) is negatively correlated with the humoral immune response of said subject.

In some embodiments, the said method is performed in vitro or ex vivo.

In particular embodiments the prognostic method of the invention comprising the step of comparing said level of one or more gene expression to a control reference value wherein:

- a high level of one or more gene expression compared to said control reference value is predictive that said subject is a high risk to be a “low responder” to said vaccine

- a low level of one or more gene expression compared to said control reference value is predictive that said subject is a high risk to be a “high responder” to said vaccine

The term “subject” as used herein refers to a mammalian, such as a rodent (e.g. a mouse or a rat), a feline, a canine or a primate. In a preferred embodiment, said subject is a human subject.

The subject according to the invention can be a healthy subject or a subject suffering from a given disease such as infectious disease prior to the vaccination of said subject with an antigen.

As used herein, the term "infectious disease" refers to a condition in which an infectious organism or agent is present in a detectable amount in the blood or in a normally sterile tissue or normally sterile compartment of a subject. Infectious organisms and agents include viruses, mycobacteria, bacteria, fungi, and parasites. The terms encompass both acute and chronic infections, as well as sepsis. In a particular embodiment the infectious organism is a virus or a bacteria that is responsible of infection such as:

Example of viral infections: Adenoviridae (ie Adenovirus); Herpesviridae (Herpes simplex, type 1, Herpes simplex, type 2, Varicella-zoster virus, Epstein-Barr virus, Human cytomegalovirus, Human herpesvirus, type 8); Papillomaviridae (Human papillomavirus); Polyomaviridae (BK virus, JC virus); Poxviridae (Smallpox); Hepadnaviridae (Hepatitis B virus); Parvoviridae (Parvovirus Bl 9); Astroviridae (Human astrovirus) Caliciviridae (Norwalk virus); Picornaviridae (coxsackievirus, hepatitis A virus, poliovirus rhinovirus) ;Coronaviridae (Severe acute respiratory syndrome-related coronavirus, strains: Severe acute respiratory syndrome virus, Severe acute respiratory syndrome coronavirus 2); Flaviviridae (Hepatitis C virus, yellow fever virus, dengue virus, West Nile virus, TBE virus, Zika virus); Matonaviridae (Rubella virus); Hepeviridae (Hepatitis E virus); Retroviridae (Human immunodeficiency virus (HIV)); Orthomyxoviridae (Influenza virus); Arenaviridae (Lassa virus); Bunyaviridae (Crimean-Congo hemorrhagic fever virus, Hantaan virus); Filoviridae (Ebola virus, Marburg virus); Paramyxoviridae (Measles virus, Mumps virus, Parainfluenza virus); Pneumoviridae (Respiratory syncytial virus); Rhabdoviridae (Rabies virus) ; Unassigned (Hepatitis D); Reoviridae (Rotavirus, Orbivirus, Coltivirus, Banna virus)

In a particular embodiment the viral infection is, Coronaviridae (Severe acute respiratory syndrome-related coronavirus, strains: Severe acute respiratory syndrome virus, Severe acute respiratory syndrome coronavirus 2) and Retroviridae (Human immunodeficiency virus (HIV)).

In a particular embodiment, the viral infection is Human immunodeficiency virus (HIV) and the vaccination with a MVA-B vaccine.

The term “MVA-B” or “Modified Vaccinia Ankara B” refers to is a particular HIV vaccine created to give immune resistance to infection by the human immunodeficiency virus. MVA-B was developed by the Spanish National Research Council's Biotechnology National Centre headed by Dr Mariano Esteban. The vaccine is based on the Modified vaccinia Ankara (MV A) virus used during the 1970s to help eradicate the smallpox virus. The B in the name "refers to HIV-B, the most common HIV subtype in Europe. The Vaccinia viruses is re- engineered to express foreign genes which are vectors for production of recombinant proteins, the most common being a vaccine delivery system for antigens (Pavot V, et al (2017). Recombinant Virus Vaccines. Methods in Molecular Biology. 1581. Springer New York. pp. 97-119). The use of MVA as a recombinant HIV vaccine (MVA-B) is being tested in approximately 300 volunteers in several Phase I studies conducted by the International AIDS Vaccine Initiative. Studies in mice and nonhuman primates have further demonstrated the safety of MVA under conditions of immune suppression. Compared to replicating vaccinia viruses, MVA provides similar or higher levels of recombinant gene expression even in non- permissive cells.

As used herein the term “humoral immune response” means the immune response involving the transformation of B cells into plasma cells that produce and secrete antibodies to a specific antigen. In the context of the present invention “assessing a subject’s humoral immune response to a vaccine” means detecting antibody response, in particular neutralizing antibody response, after immunization with an antigen, against a pathogen (infectious agent, or tumor cells). Humoral immunity prevents infection and can be of different kinds. It is characterized, for example, by neutralizing antibodies responses induced after vaccination. These functional antibodies are the main actors of protective immunity, they are able to neutralize the ability of the virus to penetrate or replicate in cells. Antibodies can belong to different immunoglobulin classes and subclasses including for example serum IgG and mucosal IgG and IgA. These Ig can also act in other ways by opsonization or complement activation, involving serum immune regulatory proteins for pathogen elimination. Other ways to defend oneself is antibody-dependent cell-mediated virus inhibition (ADCVI) and antibody-dependent cell-mediated-cytotoxicity (ADCC). This is one of the mechanisms by which antibodies act to limit and contain infection. It’s carried out by natural killer cells (NK) through different proteins: perforins (pore-forming proteins), granzymes (serine proteases), reactive oxygen intermediates, and cytokines. These cells lysis an infected cell labeled with antibody bound to antigen present on the membrane.

The term "protective humoral immunity" as used herein, means a humoral immune response that confers the essential component of protection against a pathogen.

The term "sterilizing humoral immunity" as used herein, means a humoral immune response that prevents the establishment of any detectable infection by a pathogen.

The term "long-lasting humoral immunity" as used herein, means that some aspect of humoral immunity is detectable three months after antigen administration, such as, for example, antibodies elicited by the antigen. Suitable methods of antibody detection include, but are not limited to, such methods as ELISA, immunofluorescence (IF A), focus reduction neutralization tests (FRNT), immunoprecipitation, and Western blotting.

The term "neutralizing humoral response" as used herein, means that the antibodies elicited during humoral immunity directly block the ability of a pathogen to infect cells.

The term "quick response" as used herein, means that protective humoral immunity is conferred within three weeks of antigen administration. The term "very quick response" as used herein, means that protective humoral immunity is conferred within one week of antigen administration.

A “responder to a vaccine” means that after vaccination of a subject, the serum antibody titer against the targeted antigen of said subject is sufficient to adequately clear the disease (infectious disease or tumor) and exhibit an efficient immune humoral activity. In such a case, then one can conclude that said patient has a greater proportion of immune B cells which induce an efficient immune humoral response in particular a neutralizing antibody response. Tests for determining the serum antibody titer are well known to the person skilled in the art. For example, a GFP Neutralizing assay (as described in example section) to detected an anti-antigen neutralizing activity in a serum of subjects collected 8 days after vaccination based on GFP detection by flow cytometry can be used.

Another example, according to the US FDA Guidance for Industry document, having a hemagglutinin inhibition (HI) antibody titer of 1 :40 against influenza (seroprotection) or a fourfold increase in HI titer following influenza vaccination (sero-conversion) are considered protective.

A "vaccine composition" or a “vaccine”, once it has been administered to a subject or an animal, elicits a protective immune response against said one or more antigen(s) which is (are) comprised herein. Accordingly, the vaccine composition according to the present invention, once it has been administered to the subject or the animal, induces a protective immune response against, for example, a microorganism, or to efficaciously protect the subject or the animal against infection.

A variety of substances can be used as antigens in a compound or formulation, of immunogenic or vaccine type. For example, attenuated and inactivated viral and bacterial pathogens, purified macromolecules, polysaccharides, toxoids, recombinant antigens, organisms containing a foreign gene from a pathogen, synthetic peptides, polynucleic acids (DNA, RNA), antibodies and tumor cells can be used to prepare (i) an immunogenic composition useful to induce an immune response in an individual or (ii) a vaccine useful for treating a pathological condition.

An antigen is additionally capable of being recognized by the immune system and/or being capable of inducing a humoral immune response and/or cellular immune response leading to the activation of B- and/or T-lymphocytes. An antigen can have one or more epitopes or antigenic sites (B- and T- epitopes)

Therefore, a wide variety of antigens to produce a vaccine composition useful for inducing an immune response in an individual can be used.

Those skilled in the art will be able to select an antigen appropriate for treating a particular pathological condition and will know how to determine whether an isolated antigen is favored in a particular vaccine formulation.

An isolated antigen can be prepared using a variety of methods well known in the art. A gene encoding any immunogenic polypeptide can be isolated and cloned, for example, in bacterial, yeast, insect, reptile or mammalian cells using recombinant methods well known in the art and described, for example in Sambrook et al., Molecular cloning: A Laboratory Manual, Cold Spring Harbor Laboratory, New York (1992) and in Ansubel et al., Current Protocols in Molecular Biology, John Wiley and Sons, Baltimore, MD (1998). A number of genes encoding surface antigens from viral, bacterial and protozoan pathogens have been successfully cloned, expressed and used as antigens for vaccine development. For example, the major surface antigen of hepatitis B virus, HbsAg, the P subunit of choleratoxin, the enterotoxin of E. coh. the circumsporozoite protein of the malaria parasite, and a glycoprotein membrane antigen from Epstein-Barr virus, as well as tumor cell antigens, have been expressed in various well known vector/host systems, purified and used in vaccines.

A pathologically aberrant cell may also be used in a vaccine composition according to the invention can be obtained from any source such as one or more individuals having a pathological condition or ex vivo or in vitro cultured cells obtained from one or more such individuals, including a specific individual to be treated with the resulting vaccine.

As used herein, the term “sample “ or "biological sample" as used herein refers to any biological sample of a subject and can include, by way of example and not limitation, bodily fluids and/or tissue extracts such as homogenates or solubilized tissue obtained from a subject. Tissue extracts are obtained routinely from tissue biopsy. In a particular embodiment regarding the prognostic method according to the invention, the biological sample is a body fluid sample (such blood sample) or tissue biopsy of said subject.

In preferred embodiments, the body fluid sample is a blood sample. The term “blood sample” means a whole blood sample obtained from a subject (e.g. an individual for which it is interesting to determine whether a gene expression level can be identified).

The term “prior to the administration of (said) vaccine” means that the level of one or more gene expression level selected from a group of gene consisting of: IGLV8-61, BLK, EBF1 and IFNal6 gene, is measured at least 30, 29, 28, 27, 26, 25, 24, 23, 22, 21, 20, 19, 18, 17, 16, 15, 14, 13, 12, 11, 10, 9, 8, 7, 6, 5, 4, 3, 2, 1, 0 days prior the administration of the vaccine. Preferably the level of one or more gene expression level selected from a group of gene consisting of: IGLV8-61, BLK, EBF1 and IFNal6 gene, is measured at least 15, 14, 13, 12, 11, 10, 9, 8, 7, 6, 5, 4, 3, 2, 1, 0 days prior the administration of the vaccine.

As used herein, the term " IFNal6" also known as “Interferon alpha-16” or “IFN alpha 16”, has its general meaning in the art and refers to a cytokine that in humans is encoded by the IFNA16 gene (Gene ID: 3449). Produced by macrophages, IFN-alpha 16 have antiviral activities. Interferon alpha 16 stimulates the production of two enzymes: a protein kinase and an oligoadenylate synthetase. The sequence of said protein may be found with the NCBI Reference: NM_002173 and NP_002164. As used herein, the term "IGLV8-61", also known as “Immunoglobulin lambda variable 8-61” has its general meaning in the art refers to a protein that in humans is encoded by the IGLV8-61gene (gene ID 28774). V region of the variable domain of immunoglobulin light chains that participates in the antigen recognition. Immunoglobulins, also known as antibodies, are membrane-bound or secreted glycoproteins produced by B lymphocytes. The antigen binding site is formed by the variable domain of one heavy chain, together with that of its associated light chain. Thus, each immunoglobulin has two antigen binding sites with remarkable affinity for a particular antigen. The variable domains are assembled by a process called V-(D)-J rearrangement and can then be subjected to somatic hypermutations which, after exposure to antigen and selection, allow affinity maturation for a particular antigen

The sequence of said gene may be found with the NCBI Reference: NG 000002 and NC_000022 (Reference GRCh38.pl3 Primary Assembly).

As used herein, the term BLK (Tyrosine-protein kinase BLK) also known as “B lymphocyte kinase” “BLK proto-oncogene” or “Src family tyrosine kinase” has its general meaning in the art and refers to an enzyme that in humans is encoded by the B LK gene (Gene ID: 640). BLK gene encodes a nonreceptor tyrosine-kinase of the src family of proto- oncogenes that are typically involved in cell proliferation and differentiation. The protein has a role in B-cell receptor signaling and B-cell development. The protein also stimulates insulin synthesis and secretion in response to glucose and enhances the expression of several pancreatic beta-cell transcription factors.

The sequence of said protein may be found with the NCBI Reference: NM_001715/NM_001330465 (isoform 1 and isoform 2) and NP_001317394/NP_001706 (isoform 1 and isoform 2).

As used herein, the term EBF1 also known as “Early B-Cell Factor 1” “or “Transcription factor COE1” has its general meaning in the art and refers to a protein that in humans is encoded by the EBF1 gene (Gene ID: 1879). The transcription factor EBF1 controls the expression of key proteins required for B cell differentiation, signal transduction and function (Treiber, T. et al (2010). Immunity. 32 (5): 714-725.; Hagman J. et al (2012). Current Topics in Microbiology and Immunology. 356: 17-38). The crucial role of this factor is shown in the regulation of expression of SLAM family co-receptors in B-cells (Schwartz, A. et al. (2016). Biochimica et Biophysica Acta (BBA) - Gene Regulatory Mechanisms. 1859 (10): 1259-1268).

The sequence of said protein may be found with the NCBI Reference: NM_001290360 and NP_001277289 (isoform 1)/ NM_024007 and NP_076870 (isoform 2)/ NMJ82708 and NP_874367 (isoform 3)/ NM_001324101 and NP_001311030 (isoform 4)/ NM_001324103 and NP_001311032 (isoform 5)/ NM_001324106 and NP_001311035 (isoform 6)/ NM_001324107 and NP_001311036 ((isoform 7)/ NM_001324108 and NP_001311037 (isoform 8)/ NM_001324109 and NP_001311038 (isoform 9)/ NM_001324111 and NP_001311040 (isoform 10)/ NM_001364155 and NP_001351084 (isoform 11)/ NM_001364156 and NP_001351085 (isoform 13)/ NM_001364157 and NP_001351086 (isoform 14)/ NM_001364158 and NP_001351087 (isoform 15)/ NM_001364159 and NP_001351088 (isoform 16).

The inventors have found biomarkers (gene expression level selected from a group of gene) associated with subject’s humoral immune response to a vaccine and have identified 4 biomarkers which could be used separately or in combination.

Thus, in one aspect, the present invention provides an in vitro method for assessing a subject’s humoral immune response to a vaccine, comprising the step of (a) measuring in a sample obtained from said subject prior to the administration of said vaccine, the level of one or more gene expression level selected from a group of genes consisting of: IFNal6, IGLV8- 61, BLK, and EBF1 gene and b) wherein the level of one or more gene expression of step a) is negatively correlated with the humoral immune response of said subject.

In preferred embodiments, a plurality of gene expression level biomarkers (i.e., one or more than one gene expression level biomarkers) is used in the method of prognosis. In other words, the method of the invention may comprise steps of: measuring in the biological sample plurality of gene expression level biomarkers, between one, two, three; four, gene expression level biomarker selected from a group of gene consisting of: IFNal6, IGLV8-61, BLK, and EBF1 gene present in the biological sample.

In particular embodiments, the method of prognosis is performed using the four different gene expression level biomarkers including the IFNal6, IGLV8-61, BLK, and EBF1 gene.

Measuring the expression level of a gene can be performed by a variety of techniques well known in the art.

Typically, the expression level of a gene may be determined by determining the quantity of mRNA. Methods for determining the quantity of mRNA are well known in the art. For example, the nucleic acid contained in the samples (e.g., blood, cell or tissue prepared from the patient) is first extracted according to standard methods, for example using lytic enzymes or chemical solutions or extracted by nucleic-acid-binding resins following the manufacturer's instructions. The extracted mRNA is then detected by hybridization (e. g., Northern blot analysis, in situ hybridization) and/or amplification (e.g., RT-PCR).

Other methods of Amplification include ligase chain reaction (LCR), transcription- mediated amplification (TMA), strand displacement amplification (SDA) and nucleic acid sequence-based amplification (NASBA).

Nucleic acids having at least 10 nucleotides and exhibiting sequence complementarity or homology to the mRNA of interest herein find utility as hybridization probes or amplification primers. It is understood that such nucleic acids need not be identical, but are typically at least about 80% identical to the homologous region of comparable size, more preferably 85% identical and even more preferably 90-95% identical. In certain embodiments, it will be advantageous to use nucleic acids in combination with appropriate means, such as a detectable label, for detecting hybridization.

Typically, the nucleic acid probes include one or more labels, for example to permit detection of a target nucleic acid molecule using the disclosed probes. In various applications, such as in situ hybridization procedures, a nucleic acid probe includes a label (e.g., a detectable label). A “detectable label” is a molecule or material that can be used to produce a detectable signal that indicates the presence or concentration of the probe (particularly the bound or hybridized probe) in a sample. Thus, a labeled nucleic acid molecule provides an indicator of the presence or concentration of a target nucleic acid sequence (e.g., genomic target nucleic acid sequence) (to which the labeled uniquely specific nucleic acid molecule is bound or hybridized) in a sample. A label associated with one or more nucleic acid molecules (such as a probe generated by the disclosed methods) can be detected either directly or indirectly. A label can be detected by any known or yet to be discovered mechanism including absorption, emission and/ or scattering of a photon (including radio frequency, microwave frequency, infrared frequency, visible frequency and ultra-violet frequency photons). Detectable labels include colored, fluorescent, phosphorescent and luminescent molecules and materials, catalysts (such as enzymes) that convert one substance into another substance to provide a detectable difference (such as by converting a colorless substance into a colored substance or vice versa, or by producing a precipitate or increasing sample turbidity), haptens that can be detected by antibody binding interactions, and paramagnetic and magnetic molecules or materials.

Particular examples of detectable labels include fluorescent molecules (or fluorochromes). Numerous fluorochromes are known to those of skill in the art, and can be selected, for example from Life Technologies (formerly Invitrogen), e.g., see, The Handbook — A Guide to Fluorescent Probes and Labeling Technologies). Examples of particular fluorophores that can be attached (for example, chemically conjugated) to a nucleic acid molecule (such as a uniquely specific binding region) are provided in U.S. Pat. No. 5,866, 366 to Nazarenko et al., such as 4-acetamido-4'-isothiocyanatostilbene-2,2' disulfonic acid, acridine and derivatives such as acridine and acridine isothiocyanate, 5-(2'-aminoethyl) aminonaphthalene- 1 -sulfonic acid (EDANS), 4-amino -N- [3 vinylsulfonyl)phenyl]naphthalimide-3,5 disulfonate (Lucifer Yellow VS), N-(4-anilino-l- naphthyl)maleimide, antllranilamide, Brilliant Yellow, coumarin and derivatives such as coumarin, 7-amino-4-methylcoumarin (AMC, Coumarin 120), 7-amino-4- trifluoromethylcouluarin (Coumarin 151); cyanosine; 4',6-diarninidino-2-phenylindole (DAPI); 5',5"dibromopyrogallol-sulfonephthalein (Bromopyrogallol Red); 7 -diethylamino -3 (4'-isothiocyanatophenyl)-4-methylcoumarin; diethylenetriamine pentaacetate; 4,4'- diisothiocyanatodihydro-stilbene-2,2'-disulfonic acid; 4,4'-diisothiocyanatostilbene-2,2'- disulforlic acid; 5-[dimethylamino] naphthalene- 1 -sulfonyl chloride (DNS, dansyl chloride); 4-(4'-dimethylaminophenylazo)benzoic acid (DABCYL); 4-dimethylaminophenylazophenyl- 4'-isothiocyanate (DABITC); eosin and derivatives such as eosin and eosin isothiocyanate; erythrosin and derivatives such as erythrosin B and erythrosin isothiocyanate; ethidium; fluorescein and derivatives such as 5-carboxyfluorescein (FAM), 5-(4,6diclllorotriazin-2- yDaminofluorescein (DTAF), 2'7'dimethoxy-4'5'-dichloro-6-carboxyfluorescein (JOE), fluorescein, fluorescein isothiocyanate (FITC), and QFITC Q(RITC); 2',7'-difluorofluorescein (OREGON GREEN®); fluorescamine; IR144; IR1446; Malachite Green isothiocyanate; 4- methylumbelliferone; ortho cresolphthalein; nitrotyrosine; pararosaniline; Phenol Red; B- phycoerythrin; o-phthaldialdehyde; pyrene and derivatives such as pyrene, pyrene butyrate and succinimidyl 1 -pyrene butyrate; Reactive Red 4 (Cibacron Brilliant Red 3B-A); rhodamine and derivatives such as 6-carboxy-X-rhodamine (ROX), 6-carboxyrhodamine (R6G), lissamine rhodamine B sulfonyl chloride, rhodamine (Rhod), rhodamine B, rhodamine 123, rhodamine X isothiocyanate, rhodamine green, sulforhodamine B, sulforhodamine 101 and sulfonyl chloride derivative of sulforhodamine 101 (Texas Red); N,N,N',N' -tetramethyl - 6-carboxyrhodamine (TAMRA); tetramethyl rhodamine; tetramethyl rhodamine isothiocyanate (TRITC); riboflavin; rosolic acid and terbium chelate derivatives. Other suitable fluorophores include thiol -reactive europium chelates which emit at approximately 617 mn (Heyduk and Heyduk, Analyt. Biochem. 248:216-27, 1997; J. Biol. Chem. 274:3315- 22, 1999), as well as GFP, LissamineTM, di ethylaminocoumarin, fluorescein chlorotriazinyl, naphthofluorescein, 4,7-dichlororhodamine and xanthene (as described in U.S. Pat. No. 5,800,996 to Lee et al.) and derivatives thereof. Other fluorophores known to those skilled in the art can also be used, for example those available from Life Technologies (Invitrogen; Molecular Probes (Eugene, Oreg.)) and including the ALEXA FLUOR® series of dyes (for example, as described in U.S. Pat. Nos. 5,696,157, 6, 130, 101 and 6,716,979), the BODIPY series of dyes (dipyrrometheneboron difluoride dyes, for example as described in U.S. Pat. Nos. 4,774,339, 5,187,288, 5,248,782, 5,274,113, 5,338,854, 5,451,663 and 5,433,896), Cascade Blue (an amine reactive derivative of the sulfonated pyrene described in U.S. Pat. No. 5,132,432) and Marina Blue (U.S. Pat. No. 5,830,912).

In addition to the fluorochromes described above, a fluorescent label can be a fluorescent nanoparticle, such as a semiconductor nanocrystal, e.g., a QUANTUM DOTTM (obtained, for example, from Life Technologies (QuantumDot Corp, Invitrogen Nanocrystal Technologies, Eugene, Oreg.); see also, U.S. Pat. Nos. 6,815,064; 6,682,596; and 6,649, 138). Semiconductor nanocrystals are microscopic particles having size-dependent optical and/or electrical properties. When semiconductor nanocrystals are illuminated with a primary energy source, a secondary emission of energy occurs of a frequency that corresponds to the handgap of the semiconductor material used in the semiconductor nanocrystal. This emission can he detected as colored light of a specific wavelength or fluorescence. Semiconductor nanocrystals with different spectral characteristics are described in e.g., U.S. Pat. No. 6,602,671. Semiconductor nanocrystals that can he coupled to a variety of biological molecules (including dNTPs and/or nucleic acids) or substrates by techniques described in, for example, Bruchez et al., Science 281 :20132016, 1998; Chan et al., Science 281 :2016- 2018, 1998; and U.S. Pat. No. 6,274,323. Formation of semiconductor nanocrystals of various compositions are disclosed in, e.g., U.S. Pat. Nos. 6,927, 069; 6,914,256; 6,855,202; 6,709,929; 6,689,338; 6,500,622; 6,306,736; 6,225,198; 6,207,392; 6,114,038; 6,048,616; 5,990,479; 5,690,807; 5,571,018; 5,505,928; 5,262,357 and in U.S. Patent Publication No. 2003/0165951 as well as PCT Publication No. 99/26299 (published May 27, 1999). Separate populations of semiconductor nanocrystals can he produced that are identifiable based on their different spectral characteristics. For example, semiconductor nanocrystals can he produced that emit light of different colors based on their composition, size or size and composition. For example, quantum dots that emit light at different wavelengths based on size (565 mn, 655 mn, 705 mn, or 800 mn emission wavelengths), which are suitable as fluorescent labels in the probes disclosed herein are available from Life Technologies (Carlshad, Calif.). Additional labels include, for example, radioisotopes (such as ³ H), metal chelates such as DOTA and DPTA chelates of radioactive or paramagnetic metal ions like Gd3+, and liposomes.

Detectable labels that can he used with nucleic acid molecules also include enzymes, for example horseradish peroxidase, alkaline phosphatase, acid phosphatase, glucose oxidase, beta-galactosidase, beta-glucuronidase, or beta-lactamase.

Alternatively, an enzyme can he used in a metallographic detection scheme. For example, silver in situ hybridization (SISH) procedures involve metallographic detection schemes for identification and localization of a hybridized genomic target nucleic acid sequence. Metallographic detection methods include using an enzyme, such as alkaline phosphatase, in combination with a water-soluble metal ion and a redox-inactive substrate of the enzyme. The substrate is converted to a redox-active agent by the enzyme, and the redoxactive agent reduces the metal ion, causing it to form a detectable precipitate. (See, for example, U.S. Patent Application Publication No. 2005/0100976, PCT Publication No. 2005/ 003777 and U.S. Patent Application Publication No. 2004/ 0265922). Metallographic detection methods also include using an oxido-reductase enzyme (such as horseradish peroxidase) along with a water-soluble metal ion, an oxidizing agent and a reducing agent, again to form a detectable precipitate. (See, for example, U.S. Pat. No. 6,670,113).

Probes made using the disclosed methods can be used for nucleic acid detection, such as ISH procedures (for example, fluorescence in situ hybridization (FISH), chromogenic in situ hybridization (CISH) and silver in situ hybridization (SISH)) or comparative genomic hybridization (CGH).

In situ hybridization (ISH) involves contacting a sample containing target nucleic acid sequence (e.g., genomic target nucleic acid sequence) in the context of a metaphase or interphase chromosome preparation (such as a cell or tissue sample mounted on a slide) with a labeled probe specifically hybridizable or specific for the target nucleic acid sequence (e.g., genomic target nucleic acid sequence). The slides are optionally pretreated, e.g., to remove paraffin or other materials that can interfere with uniform hybridization. The sample and the probe are both treated, for example by heating to denature the double stranded nucleic acids. The probe (formulated in a suitable hybridization buffer) and the sample are combined, under conditions and for sufficient time to permit hybridization to occur (typically to reach equilibrium). The chromosome preparation is washed to remove excess probe, and detection of specific labeling of the chromosome target is performed using standard techniques. For example, a biotinylated probe can be detected using fluorescein-labeled avidin or avidin-alkaline phosphatase. For fluorochrome detection, the fluorochrome can be detected directly, or the samples can be incubated, for example, with fluorescein isothiocyanate (FITC)-conjugated avidin. Amplification of the FITC signal can be affected, if necessary, by incubation with biotin-conjugated goat anti-avidin antibodies, washing and a second incubation with FITC-conjugated avidin. For detection by enzyme activity, samples can be incubated, for example, with streptavidin, washed, incubated with biotin-conjugated alkaline phosphatase, washed again and pre-equilibrated (e.g., in alkaline phosphatase (AP) buffer). For a general description of in situ hybridization procedures, see, e.g., U.S. Pat. No. 4,888,278.

Numerous procedures for FISH, CISH, and SISH are known in the art. For example, procedures for performing FISH are described in U.S. Pat. Nos. 5,447,841; 5,472,842; and 5,427,932; and for example, in Pirlkel et al., Proc. Natl. Acad. Sci. 83:2934-2938, 1986; Pinkel et al., Proc. Natl. Acad. Sci. 85:9138-9142, 1988; and Lichter et al., Proc. Natl. Acad. Sci. 85:9664-9668, 1988. CISH is described in, e.g., Tanner et al., Am. .1. Pathol. 157: 1467- 1472, 2000 and U.S. Pat. No. 6,942,970. Additional detection methods are provided in U.S. Pat. No. 6,280,929.

Numerous reagents and detection schemes can be employed in conjunction with FISH, CISH, and SISH procedures to improve sensitivity, resolution, or other desirable properties. As discussed above probes labeled with fluorophores (including fluorescent dyes and QUANTUM DOTS®) can be directly optically detected when performing FISH. Alternatively, the probe can be labeled with a nonfluorescent molecule, such as a hapten (such as the following non-limiting examples: biotin, digoxigenin, DNP, and various oxazoles, pyrrazoles, thiazoles, nitroaryls, benzofurazans, triterpenes, ureas, thioureas, rotenones, coumarin, courmarin-based compounds, Podophyllotoxin, Podophyllotoxin-based compounds, and combinations thereof), ligand or other indirectly detectable moiety. Probes labeled with such non-fluorescent molecules (and the target nucleic acid sequences to which they bind) can then be detected by contacting the sample (e.g., the cell or tissue sample to which the probe is bound) with a labeled detection reagent, such as an antibody (or receptor, or other specific binding partner) specific for the chosen hapten or ligand. The detection reagent can be labeled with a fluorophore (e.g., QUANTUM DOT®) or with another indirectly detectable moiety, or can be contacted with one or more additional specific binding agents (e.g., secondary or specific antibodies), which can be labeled with a fluorophore.

In other examples, the probe, or specific binding agent (such as an antibody, e.g., a primary antibody, receptor or other binding agent) is labeled with an enzyme that is capable of converting a fluorogenic or chromogenic composition into a detectable fluorescent, colored or otherwise detectable signal (e.g., as in deposition of detectable metal particles in SISH). As indicated above, the enzyme can be attached directly or indirectly via a linker to the relevant probe or detection reagent. Examples of suitable reagents (e.g., binding reagents) and chemistries (e.g., linker and attachment chemistries) are described in U.S. Patent Application Publication Nos. 2006/0246524; 2006/0246523, and 2007/ 01 17153.

It will be appreciated by those of skill in the art that by appropriately selecting labelled probe-specific binding agent pairs, multiplex detection schemes can he produced to facilitate detection of multiple target nucleic acid sequences (e.g., genomic target nucleic acid sequences) in a single assay (e.g., on a single cell or tissue sample or on more than one cell or tissue sample). For example, a first probe that corresponds to a first target sequence can he labelled with a first hapten, such as biotin, while a second probe that corresponds to a second target sequence can be labelled with a second hapten, such as DNP. Following exposure of the sample to the probes, the bound probes can he detected by contacting the sample with a first specific binding agent (in this case avidin labelled with a first fluorophore, for example, a first spectrally distinct QUANTUM DOT®, e.g., that emits at 585 mn) and a second specific binding agent (in this case an anti-DNP antibody, or antibody fragment, labelled with a second fluorophore (for example, a second spectrally distinct QUANTUM DOT®, e.g., that emits at 705 mn). Additional probes/binding agent pairs can be added to the multiplex detection scheme using other spectrally distinct fluorophores. Numerous variations of direct, and indirect (one step, two step or more) can he envisioned, all of which are suitable in the context of the disclosed probes and assays.

Probes typically comprise single-stranded nucleic acids of between 10 to 1000 nucleotides in length, for instance of between 10 and 800, more preferably of between 15 and 700, typically of between 20 and 500. Primers typically are shorter single-stranded nucleic acids, of between 10 to 25 nucleotides in length, designed to perfectly or almost perfectly match a nucleic acid of interest, to be amplified. The probes and primers are “specific” to the nucleic acids they hybridize to, i.e. they preferably hybridize under high stringency hybridization conditions (corresponding to the highest melting temperature Tm, e.g., 50 % formamide, 5x or 6x SCC. SCC is a 0.15 M NaCl, 0.015 M Na-citrate).

The nucleic acid primers or probes used in the above amplification and detection method may be assembled as a kit. Such a kit includes consensus primers and molecular probes. A preferred kit also includes the components necessary to determine if amplification has occurred. The kit may also include, for example, PCR buffers and enzymes; positive control sequences, reaction control primers; and instructions for amplifying and detecting the specific sequences.

In a particular embodiment, the methods of the invention comprise the steps of providing total RNAs extracted from blood and subjecting the RNAs to amplification and hybridization to specific probes, more particularly by means of a quantitative or semi- quantitative RT-PCR.

In another preferred embodiment, the expression level is determined by DNA chip analysis. Such DNA chip or nucleic acid microarray consists of different nucleic acid probes that are chemically attached to a substrate, which can be a microchip, a glass slide or a microsphere-sized bead. A microchip may be constituted of polymers, plastics, resins, polysaccharides, silica or silica-based materials, carbon, metals, inorganic glasses, or nitrocellulose. Probes comprise nucleic acids such as cDNAs or oligonucleotides that may be about 10 to about 60 base pairs. To determine the expression level, a sample from a test subject, optionally first subjected to a reverse transcription, is labelled and contacted with the microarray in hybridization conditions, leading to the formation of complexes between target nucleic acids that are complementary to probe sequences attached to the microarray surface. The labelled hybridized complexes are then detected and can be quantified or semi-quantified. Labelling may be achieved by various methods, e.g. by using radioactive or fluorescent labelling. Many variants of the microarray hybridization technology are available to the man skilled in the art (see e.g. the review by Hoheisel, Nature Reviews, Genetics, 2006, 7:200- 210).

Expression level of a gene may be expressed as absolute expression level or normalized expression level. Typically, expression levels are normalized by correcting the absolute expression level of a gene by comparing its expression to the expression of a gene that is not a relevant for determining the cancer stage of the patient, e.g., a housekeeping gene that is constitutively expressed. Suitable genes for normalization include housekeeping genes such as the actin gene ACTB, ribosomal 18S gene, GUSB, PGK1, TBP, HPRT1 and TFRC. TATA-binding protein TBP) and hypoxanthine phosphoribosyl transferase 1 (HPRTT) were used as reference genes in the present study. This normalization allows the comparison of the expression level in one sample, e.g., a patient sample, to another sample, or between samples from different sources.

The man skilled in the art also understands that the same technique of assessment of the expression level of a gene should of course be used for obtaining the reference value and thereafter for assessment of the expression level of a gene of a patient subjected to the method of the invention.

Said reference control values may be determined in regard to the level of gene expression biomarker present in blood samples taken from one or more healthy subject(s) or in a control population.

In one embodiment, the method according to the present invention comprises the step of comparing said level of humoral immune response to a vaccine -specific gene expression level biomarkers (“Biomarkerl”: IFNal6 gene and/or “Biomarker2”: IGLV8-61 gene and/or “Biomarker3”: BLK gene and/or “Biomarker4”: EBF1 gene) to a control reference value wherein a high level of “humoral immune response to a vaccine -specific” gene expression biomarkers (“Biomarkerl”: IFNal6 gene and/or “Biomarker2”: IGLV8-61 gene and/or “Biomarker3”: BLK gene and/or “Biomarker4”: EBF1 gene) compared to said control reference value is predictive of a high risk to be a “low responder” to said vaccine and a low “"humoral immune response to a vaccine -specific” gene expression biomarkers (Biomarkerl”: IFNal6 gene and/or “Biomarker2”: IGLV8-61 gene and/or “Biomarker3”: BLK gene and/or “Biomarker4”: EBF1 gene) compared to said control reference value is predictive of a high risk to be a “high responder” to said vaccine.

The control reference value may depend on various parameters such as the method used to measure the level humoral immune response to a vaccine -specific gene expression level biomarkers (“Biomarkerl”: IFNal6 gene and/or “Biomarker2”: IGLV8-61 gene and/or “Biomarker3”: BLK gene and/or “Biomarker4”: EBF1 gene) or the gender of the subject.

Control reference values are easily determinable by the one skilled in the art, by using the same techniques as for determining the level of gene expression biomarker in a blood samples previously collected from the patient under testing.

A “reference value” can be a “threshold value” or a “cut-off value”. Typically, a "threshold value" or "cut-off value" can be determined experimentally, empirically, or theoretically. A threshold value can also be arbitrarily selected based upon the existing experimental and/or clinical conditions, as would be recognized by a person of ordinary skilled in the art. The threshold value has to be determined in order to obtain the optimal sensitivity and specificity according to the function of the test and the benefit/risk balance (clinical consequences of false positive and false negative). Typically, the optimal sensitivity and specificity (and so the threshold value) can be determined using a Receiver Operating Characteristic (ROC) curve based on experimental data. Preferably, the person skilled in the art may compare the level of gene expression biomarkers (“Biomarkerl”: IFNal6 gene and/or “Biomarker2”: IGLV8-61 gene and/or “Biomarker3”: BLK gene and/or “Biomarker4”: EBF1 gene) with a defined threshold value. In one embodiment of the present invention, the threshold value is derived from the gene expression level (or ratio, or score) determined in a blood sample derived from one or more subjects who are responders (to the method according to the invention). In one embodiment of the present invention, the threshold value may also be derived from gene expression level (or ratio, or score) determined in a blood sample derived from one or more subjects or who are non-responders. Furthermore, retrospective measurement of the gene expression level (or ratio, or scores) in properly banked historical subject samples may be used in establishing these threshold values.

For instance in the MVA-B vaccination study and with the expression level of IFNal6, IGLV8-61, BLK, and/or EBF1 gene assessed by microarray hybridation, the ROC curve analysis revealed that choosing a cut-off value at 3.5 unit expression of IFNal6 mRNA, and/or at 6.5 unit expression of IGLV8-61 mRNA and/or at 6 unit expression of BLK, and/or at 5.9 unit expression of EBF1 mRNA levels allowed to effectively discriminate responders from / non responders blood sample and which could be used as predetermined reference level for IFNal6, IGLV8-61, BLK, and/or EBF1.

"Risk" in the context of the present invention, relates to the probability that an event will occur over a specific time period, as in humoral immune response of a subject to a vaccine, and can mean a subject's "absolute" risk or "relative" risk. Absolute risk can be measured with reference to either actual observation post-measurement for the relevant time cohort, or with reference to index values developed from statistically valid historical cohorts that have been followed for the relevant time period. Relative risk refers to the ratio of absolute risks of a subject compared either to the absolute risks of low-risk cohorts or an average population risk, which can vary by how clinical risk factors are assessed. Odds ratios, the proportion of positive events to negative events for a given test result, are also commonly used (odds are according to the formula p/(l-p) where p is the probability of event and (1- p) is the probability of no event) to no conversion. Alternative continuous measures, which may be assessed in the context of the present invention, include time to humoral immune response of a subject to a vaccine risk reduction ratios.

"Risk evaluation," or "evaluation of risk" in the context of the present invention encompasses making a prediction of the probability, odds, or likelihood that an event (humoral immune response of a subject to a vaccine) may occur, the rate of occurrence of the event or conversion from one state to another, i.e., from a “high responder” to said vaccine to “low responder” to said vaccine. Risk evaluation can also comprise prediction of future clinical parameters, traditional laboratory risk factor values, or other indices of “humoral response”, such as cellular population determination in peripheral tissues, in serum or other fluid, either in absolute or relative terms in reference to a previously measured population. The methods of the present invention may be used to make continuous or categorical measurements of the risk of an event (humoral immune response of a subject to a vaccine), thus prognosing and defining the risk spectrum of a category of subjects defined as being “high responder” to a vaccine. In the categorical scenario, the invention can be used to discriminate between normal and other subject cohorts at higher risk to be “high responder” to said vaccine. In other embodiments, the present invention may be used so as to help to discriminate those having “high responder” to a vaccine from “low responder” to said vaccine

Typically, the methods for assessing a subject’s humoral immune response to a vaccine may further comprise measuring, in a biological sample obtained from the subject, the level Eubacterium in stool sample and Prevotella in skin sample.

In a particular embodiment the level Eubacterium in stool sample and Prevotella in skin sample is performed by using 16S ribosomal sequencing.

Kit for performing the method of the invention

A further object of the invention relates to kits for performing the methods of the invention, wherein said kits comprise means for measuring the expression level of one or more gene expression level selected from a group of gene consisting of: IGLV8-61, BLK, EBF1 and IFNal6 gene of the invention in the sample obtained from the patient for use to assess a subject’s humoral immune response to a vaccine.

Accordingly, the present invention also relates to a kit of the invention comprising means for determining the expression level of one or more gene expression level selected from a group of gene consisting of IGLV8-61, BLK, EBF1 and IFNal6 gene.

In one embodiment, the present invention relates to a kit for use to assess a subject’s humoral immune response to a vaccine, comprising:

- at least a means for determining the expression level of one or more gene expression level selected from a group of gene consisting of IGLV8-61, BLK, EBF1 and IFNal6 gene and

- instructions for use.

In a particular embodiment, the kit for use comprising: - amplification primers and/or probe for determining the expression level one or more gene expression level selected from a group of gene consisting of IGLV8-61, BLK, EBF1 and IFNal6 gene,

- instructions for use.

The kits may include probes, primers macroarrays or microarrays as above described. For example, the kit may comprise a set of probes as above defined, usually made of DNA, and that may be pre-labelled. Alternatively, probes may be unlabelled and the ingredients for labelling may be included in the kit in separate containers. The kit may further comprise hybridization reagents or other suitably packaged reagents and materials needed for the particular hybridization protocol, including solid-phase matrices, if applicable, and standards. Alternatively the kit of the invention may comprise amplification primers that may be pre- labelled or may contain an affinity purification or attachment moiety. The kit may further comprise amplification reagents and also other suitably packaged reagents and materials needed for the particular amplification protocol.

Therapeutic method

The invention also relates to a method for vaccinating a subject in need thereof with an antigen wherein, prior to vaccination; the level of one or more gene expression level selected from a group of gene consisting of IFNal6, IGLV8-61, BLK and EBF1 gene obtained from said subject have been detected by one of method of the invention.

Another object of the present invention is a method for vaccinating in a subject comprising, prior to vaccination the steps of a) providing a blood sample from a subject, b) detecting the level of one or more gene expression level selected from a group of gene consisting of IFNal6, IGLV8-61, BLK and EBF1 gene obtained from said subject c) comparing the level determined in step b) with a reference value and if level determined at step b) is lower than the reference value, vaccinating the subject with antigen.

The invention will be further illustrated by the following figures and examples. However, these examples and figures should not be interpreted in any way as limiting the scope of the present invention. FIGURES:

Figure 1: Blood gene expression combined with host microbiota before vaccination shapes MVA-B responses

A. Investigation of the blood gene expression (w-2, wO) correlated with MVA-Nab response (w8) and host genus diversity (wO), for skin and stool. The Spearman correlation test was applied with P<0.05 defined as statistically significant. B. Table shows the significant correlation coefficients and P-values for each of the three genes with the abundance of the genus correlated with MVA-Nab response. C. ROC curves show the specificity and the sensitivity of the logistic regression models, i.e., the proportion of correctly predicted responders and nonresponders, respectively. The logistic regression is based on the expression of the minimal gene signature (IGLV8, EBF1, and BLK) and the abundance of Eubacterium and Prevotella, respectively, in stool and skin.

Figure 2: ROC gene Nab: IFNal6, IGLV8-61, BLK and EBF1

Among 154 genes associated with NAb (Figure 1A), we found that IFNal6 was associated with B cell differentiation and proliferation. ROC curves show the specificity and the sensitivity of the logistic regression models, i.e., the proportion of correctly predicted responders and nonresponders, respectively. A. The logistic regression based on single level of IGLV8, B. EBF1, C. BLK and D. IFNal6. E. The logistic regression is based on the expression of 4 genes IGLV8, EBF1, IFNal6 and BLK.

EXAMPLE:

Methods:

Skin and feces sampling

For each individual, skin swab samples from the deltoid muscle region (-5-20 cm below the vaccine administration site) were collected before the vaccination (wO). Skin samples were collected with Catch-All™ Sample Collection Swab kits moistened with SCF-1 solution. The skin surface was sampled for 30 seconds by firmly swabbing the cotton tip back and forth -50 times. The cotton tip was stored in sterile tubes with MoBio solution at -80°C until DNA extraction. Fecal samples for each participant were collected in sterile fecal collection tubes the day before the vaccination, matching the skin sample time points. All samples were stored at 4-5°C until their reception at the IMP ACTA clinical trial site, where they were cryopreserved at -80°C. All samples were shipped on dry ice to the IrsiCaixa AIDS Research Institute for DNA extraction, amplification, and sequencing. DNA extraction and amplicon sequencing from skin and fecal samples

DNA extraction was performed with the DNA Extraction kit from Epicentre Technologies© (Madison, WI, USA). Six aliquots of buffer solution from the DNA extraction kit were used as negative controls. To amplify the variable V3-V4 region from the 16S rRNA gene, we used the primer pair described in the MiSeq™ rRNA Amplicon Sequencing protocol, which already has the Illumina adapter overhang nucleotide sequences added to the 16S rRNA V3-V4-specific primers, i.e., 16S F 5’-(TCG GCA GCG TCA GAT GTG TAT AAG AGA CAG CCT ACG GGN GGC WGC AG)-3’(SEQ ID N°6, Table 2) and 16S R 5’-(GTC TCG TGG GCT CGG AGA TGT GTA TAA GAG ACA GGA CTA CHV GGG TAT CTA ATC C) -3’(SEQ ID N°7, Table 2). Amplifications were performed in triplicate 25-pL reactions, each containing 2.5 pL of non-diluted DNA template, 12.5 pL of KAPA HiFi HotStart Ready Mix (containing KAPA HiFi HotStart DNA Polymerase, buffer, MgC12, and dNTPs, KAPA Biosystems Inc., Wilmington, MA, USA), and 5 pL of each primer at 1 pM. Thermal cycling conditions consisted of an initial denaturation step (3 minutes at 95°C), followed by 30 cycles of denaturation (30 seconds at 95°C), annealing (30 seconds at 55°C), and extension (30 seconds at 72°C). These were followed by a final extension step of 10 minutes at 72°C. Once the desired amplicon was confirmed in 1% agarose gel electrophoresis, all three replicates were pooled and stored at -30°C until the sequencing library was prepared. After amplified DNA templates were cleaned up for non-DNA molecules and Illumina sequencing adapters and dual indices attached with the Nextera XT Index Kit (Illumina, Inc.), the corresponding PCR amplification program was run, as described in the MiSeq 16S rRNA Amplicon Sequencing protocol. After a second round of cleanup, amplicons were quantified with the Quant-iT™ PicoGreen® dsDNA Assay Kit (Invitrogen, Carlsbad, MA, USA) and diluted in equimolar concentrations (4 nM) for further pooling. Sequencing was performed on an Illumina MiSeq™ platform (Illumina, Inc.) according to the manufacturer's specifications to generate a median of 30,644 paired-end sequences of -300 bp length in each direction (-61,289 reads per sample).

Sequence quality control and microbiota analyses

The quality of MiSeq raw sequences was assessed with the FastQC software (A. and Bittencourt a, 2010) (http://www.bioinformatics.babraham.ac.uk/proiects/fastqc/). Sequences were trimmed with Trimmommatic (Bolger et al., 2014), with a cutoff value of Q30 for both ends, a minimum mean threshold of Q20 for 30-bp-sliding window across sequences, and a minimum read length of 250 bp. After quality control, 28 samples including controls (n=8) and volunteers (n=10, 5 women and 5 men) for skin and stools, were further analyzed. Mothur pipeline (Schloss et al., 2009) was used to bin 16S rDNA sequences into operational taxonomic units (OTUs) with a threshold of 97% sequence similarity. OTUs present in only a single sample were discarded. Rarefaction curves were represented by defining the maximum subsampling size as the number of sequences of the sample with the fewest sequences (2751 sequences for skin samples, and 1059 sequences for stool samples). Richness and diversity indexes were estimated by using the summary, single module implemented in mothur. For taxonomical analysis, 16S rDNA sequences were classified according to the GreenGenes database (McDonald et al., 2012) version 13.5.99.

MVA-GFP Neutralizing antibody assay

Anti-MVA neutralizing activities were evaluated in serum collected at week 8 (w8) with an assay based on GFP detection by flow cytometry (Cosma et al., 2004; Earl et al., 2003). It used HeLa cells as targets and a recombinant strain of MVA expressing the enhanced Aequoriae GFP (Garcia et al., 2011). Serial dilutions of heat inactivated serum were performed in 96-well round-bottom tissue culture plates (TPP, Zurich, Switzerland) containing DMEM (Gibco, Invitrogen) supplemented with 2% fetal calf serum (PAA, Laboratories GmbH, Pashing, Austria). MVAeGFP was then added to each well at a MOI of 0.25. The plate was then incubated for 1 hour at 37°C until the addition of 1 x 10⁵ HeLa cells. The incubation then continued for an additional 16 hours at 37°C, 0.5% CO2. After trypsinization, the cells were washed with PBS supplemented with 0.5% fetal calf serum and 2 mM EDTA and fixed with 2% formaldehyde. GFP expression was analyzed with FACSCanto II and Diva software (BD Biosciences). The percentage of neutralization was defined as the ratio of the reduction in the number of GFP-expressing cells to the number of GFP-expressing cells in untreated control wells.

RNA extraction and data preprocessing for transcriptomic analysis

Whole blood samples of 2.5 mL were collected in PAXgene RNA tubes (PreAnalytix) twice from each volunteer two weeks before (w-2) and the day of the vaccination (wO). These tubes enable the preservation and stabilization of RNA (storage at -80°C). Total RNA was extracted from whole blood according to the instructions in the handbook accompanying the PAXgene blood RNA Kit (PreAnalytiX, Hombrechtikon, Switzerland). RNA purity and integrity were assessed on the Agilent 2100 Bioanalyzer with the RNA 6000 Nano LabChip reagent set (Agilent, Palo Alto, CA, USA). Samples for microarray hybridization were prepared as described in the Affymetrix GeneChip WT PLUS Reagent Kit User Manual (Affymetrix, Inc., Santa Clara, CA, USA). For hybridization (to Affymetrix Human Gene 2.1 ST Array Plates), washing, staining, and scanning took place in an Affymetrix GeneTitan system, controlled by the Affymetrix GeneChip Command Console software w4.2. Background signal correction was performed by applying the backgroundCorrect function from the limma package on the perfect match (PM) signals with R Software 3.3.1. The underlying model is the normal-exponential convolution model from RMA (chip intensity: addition of a signal exponentially distributed, chip noise: follows Gaussian distribution) (Irizarry et al., 2003). The variance stabilizing transformation algorithm (justvsn function from the vsn package (Huber et al., 2002) was applied to the background corrected signal (monotonic transformation), and the signal then transformed back to its usual scale by exponentiation (base 2). To make the chips comparable, a quantile normalization (Bolstad et al., 2003) (normalize function from the affy package) was then applied to the variance- stabilized signal. The probe signals for replicated arrays were averaged and a quantile normalization performed anew. In all, 24,768 probes were analyzed.

Statistical analyses

Microbiome samples were clustered according to their genus composition by a nonmetric multidimensional scaling (NMDS) approach based on ecological distance matrices calculated by Bray-Curtis dissimilarities, as implemented in R (Vegan, metaMDS, and ggplot2 packages). NMDS ellipses were drawn based on a confidence interval (CI) of 0.95. To determine significant factors that describe the community structure better, we used a multivariate ADONIS test with terms added sequentially. The associations between baseline genus abundance or genus diversity, blood gene expression, and MVA-Nab response were evaluated by using the Spearman rank correlation test with significance defined by C- value <0.05. The heatmap was performed with values row-centered and scaled, Pearson correlation as the distance method and a dendrogram computed and reordered based on row means. The heatmap, logistic regression analyses, and ROC curves were performed and generated with R. Ingenuity® pathway analysis (IPA®) was used to perform functional enrichment analyses and identify new targets or candidate biomarkers within the context of biological systems. It provided the canonical pathways, molecular/cellular functions, and networks that were statistically overrepresented in the gene signatures.

Ethics and community involvement

The study was conducted in accordance with the Declaration of Helsinki and the International Conference on Harmonization Good Clinical Practice guidelines and approved by the relevant regulatory and independent ethics committees. Each participant provided written informed consent before study entry. The study was registered and approved by the Peru regulatory authorities (IMP ACTA IRB 0037-2014-CE; Peru NIH 396-2014-OG-OGITT- OPE/INS).

Sequence and data availability

The normalized microarray data that support the finding of this study have been deposited in ArrayExpress with the accession code E-MT AB-9642.

Result

Study of host microbiota before vaccination and relation to post-vaccination humoral responses

The study included five men and five women (18-45 years old) vaccinated by the intramuscular route to assess the safety and immunogenicity of MVA-HIV clade B (MVA-B), results reported elsewhere (Sanchez & Goncalves, Frontiers in Immunology in press). Exploratory analysis of whole blood samples at two distinct time-points before vaccination (w-2 and wO) studied the gene expression profile and the skin and stool samples for microbiome analysis (wO) at baseline. As expected, the microbial composition differed between the skin and stool samples. In addition, the stool samples showed dissimilarities between men and women, but this comparison did not reach statistically significant differences (P < 0.097). The predominant microbial families relatively abundant in skin samples were Moraxellaceae, Staphylococcaceae and Pseudomonadaceae, whereas Ruminococcaceae, Lachnospiraceae, Prevotellaceae, and Bacteroidaceae were predominant in stool samples. The 16S RNA sequencing generated several metrics: richness (sobs: number of observed OTUs; chao: Chaol richness estimate; ace: Abundance-based coverage estimation) and diversity (Shannon: Shannon diversity index; sd invsimpson: inverse Simpson diversity index). The amplitude of the humoral response was defined by the MVA-specific IgG neutralizing antibodies measured in serum at w8 post-vaccination. We observed no correlation between the MVA-Nab response and the baseline indexes of diversity and richness in either skin or stool (data not shown). We did however find significant positive correlations between the abundance of both skin Prevotella (r = 0.76, P = 0.0159) (data not shown) and fecal Eubacterium (r = 0.68, P = 0.0351) (data not shown) at baseline with MVA-Nab response.

Whole blood gene expression and host microbiota before vaccination are associated with post-vaccination humoral responses

To improve our understanding of host molecular mechanisms potentially associated with skin and gut microbiota that may be involved in vaccine immunogenicity, we counted the number of genes at baselines that were correlated with the MVA-Nab response at w8. We confirmed that gene expression of the baseline samples did not differ between w-2 and wO using hierarchal clustering analysis (data not shown). Out of all samples, we found 154 significant genes correlated with the MVA-Nab response (P < 0.05) (Figure 1A). However, no correlation was observed between genus diversity and MVA-Nab response.

Next, we looked for a correlation between the microbiota diversity index and the genes (n=154) correlated at baseline with MVA-Nab responses. We found 22 genes for skin and 19 for stool that were correlated with at least one diversity index (Shannon or sd invsimpson) (Figure 1A and Table 1), including 10 common genes to the skin and stool samples. Among these genes, we observed one gene cluster positively correlated with MVA-Nab response and another negatively correlated with humoral response (data not shown). According to the IPA analysis, the negatively correlated genes appear to be involved in protein transmembrane transport, translation and transcription regulation, cell division, migration, proliferation, and differentiation, as well as in the oxidation reduction and metabolic processes. The positively correlated genes, on the other hand, appeared involved in cell homeostasis and migration, cell growth, proliferation, regulation of gene expression, the apoptotic process, exocytosis, and intracellular signal transduction. Interestingly, among the 10 common genes to the skin and stool samples we found the IGLV8-61, BLK, and EBF1 genes which are involved in antigen recognition, B cell development, proliferation, and differentiation, and in the positive regulation of transcription in B cell and B cell receptor signaling (data not shown). Surprisingly these three significant genes involved in B cell development stages were negatively correlated with the baseline abundance of Prevotella and Eubacterium, respectively for skin and stool (Figure IB). To assess the predictive power of this signature of three genes and each of the two microbial genera, we ran logistic regression models (Figure 1C). Use of the expression of the three genes and Prevotella abundance in the skin microbiota has an 85.42% chance, assessed by its area under the curve, of correctly predicting MVA-Nab responders, while with the three-gene signature and Eubacterium abundance in the stool microbiota there is an 89.58% chance of correctly predicting MVA-Nab responders (Figure 1C). These results suggest that advanced B lymphocyte differentiation before vaccination, potentially signaled by high expression of these three genes, and associated with low abundance of Prevotella or Eubacterium, is associated with poor MVA-Nab response. DISCUSSION

To our knowledge, this work is the first to investigate the crosstalk between pre- vaccination host gene expression in blood cells, skin and stool microbiota and their association with the intensity of ensuing post-vaccination Nab responses. The data may provide important guidance for future design and refinement of vaccine strategies aiming at the induction of neutralizing antibody-mediated immunity. The limitation of this study is the small number of individuals included. However, the strength of our work is the availability of two sets of gene expression data collected at baseline (w-2, wO) that is often absent in other studies. It is intriguing to discover three genes, all involved in B cell differentiation and proliferation correlated with humoral responses 2 months later. Further validation studies are necessary in the future.

First, we observed that the abundance of particular skin or stool bacteria were associated with the MVA-Nab response. Abundant Prevotella in the skin at baseline was positively correlated with MVA-Nab response. Prevotella is known to promote mucosal inflammation and to stimulate production of epithelial cell cytokines (Larsen, 2017). Prevotella is also found in larger numbers in the skin of women aged 60-76 years than in that of women in their 20s and 30s and was enriched in all of the skin sites of the older group compared to the younger ones (Shibagaki et al., 2017). In stool, we found that Eubacterium abundance at baseline was positively correlated with the MVA-Nab response. This family of bacteria is known to be associated with gut health (King et al., 2018; Le Bastard et al., 2018; Rinninella et al., 2019), and several of its species are higher in centenarians than in either young or elderly adults (Biagi et al., 2010). The potential impact of the gut microbiota on vaccine immunogenicity has been already investigated with systemic vaccines (Huda et al., 2014) and with oral vaccines including those of rotavirus (RVV), polio, and cholera, mainly in infants/children living in low-income countries (Levine, 2010; Magwira and Taylor, 2018). For example, bacteria related to Streptococcus bovis species were more abundant before vaccination in Ghanaian vaccine-responders than non-responders and were positively associated with RVV efficacy, whereas Bacteroides and Prevotella species were more common in the microbiome of nonresponders and correlated with a lack of RVV response (Harris et al., 2017). In Bangladeshi infants, the pre-vaccination presence of Bifidobacterium was positively associated with some adaptive immunological responses, such as CD4⁺ and CD8⁺ T-cell proliferative responses to BCG and tetanus toxoid vaccinations as well as specific IgG responses to tetanus toxoid and hepatitis B vaccines, whereas high levels of enteric pathogens such as Enterobacteriales and Pseudomonadales were associated with neutrophilia and poorer vaccine responses (Huda et al., 2014).

Secondly, we examined the pre-vaccination host blood genes that were correlated with MVA- Nab intensity. We then investigated microbiota abundance to decipher a minimal gene signature predictive of MVA-Nab responsiveness. Interestingly, within this signature we find BLK, IGLV8-61 and EBF1 involved in B cell development, proliferation and differentiation and in the positive regulation of transcription in B cells and B cell receptor signaling. The BLK gene belongs to the family of protein tyrosine kinases src, and the B cells activation induces BLK gene product phosphorylation playing a key role in transmitting signals through surface immunoglobulins which supports the pro-B to pre-B transition and the signaling for growth arrest and apoptosis downstream of B-cell receptor (Burkhardt et al., 1991). BLK also plays a role in the development, differentiation, and activation of B cells and in the intracellular signaling pathway. BLK is detected in pro-B cells and persists in mature B cells but is absent in plasma cells. Triple protein tyrosine kinase (SFK)-deficient mice — BLK, LYN, and FYN — have impaired NFkB signaling and B cell development (Saijo et al., 2003). EBF1, an early B cell factor 1, is one of the transcription factors essential for orchestrating the development of the B cell line. Heterozygosity of EBF1 results in the deregulation of at least eight transcription factors involved in lymphopoiesis and the deregulation of key proteins that play a crucial role in the survival, development, and differentiation of pro-B cells (Musa et al., 2018). IGLV8 (variable domain) is a glycoprotein produced by B lymphocytes; its binding of a specific antigen triggers the clonal expansion and differentiation of B lymphocytes into immunoglobulin-secreting plasma cells. The link between microbiota and host blood transcriptome has also been studied previously by Nakaya et al., who showed that TLR5 expression in blood 3 days after influenza vaccination was correlated with antibody response 28 days later (Nakaya et al., 2011). This correlation was significantly lower in TLR5 -deficient mice immunized with TIV compared to wild-type mice. As influenza vaccine does not stimulate TLR5 directly, however, Oh et al. demonstrated with germ-free or antibiotic- treatment that the commensal bacteria were the source of the TLR5 ligands responsible for enhancing immune response to TIV (Oh et al., 2014). It should be noted that in our study the three genes were negatively correlated with MVA-Nab response and microbial diversity of both skin and stool samples but also with the abundance of the Prevotella family in skin and the Eubacterium family in stool. The logistic regression based on the expression of these three genes and Prevotella and Eubacterium abundance for, respectively, skin and stool, highlights the predictive power of this signature for the MVA-Nab immune responses. These results propose that an advanced differentiation state of B lymphocytes before vaccination, potentially represented by a high expression of these three genes and associated with low genus abundance and diversity, might be associated with poor MVA-Nab response.

Table 1: Skin and stool genus diversity correlations with blood gene expression

Correlation coefficient and P-value for the relations between genus diversity (shannon: diversity shannon index, sd invsimpson: inverss simpson diversity index) and the genes correlated with MVA-Nab response in a. skin and b. stool. Three genes involved in B cell function and correlated in both skin and stool are highlighted. Table 2: mRNA used in the present invention:

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Claims

CLAIMS:

1. An in vitro method for assessing a subject’s humoral immune response to a vaccine, said method, comprising the step of (a) measuring in a sample obtained from said subject prior to the administration of said vaccine, the level of one or more gene expression level selected from a group of gene consisting of: IFNal6, IGLV8-61, BLK, and EBF1 gene and b) wherein the level of one or more gene expression of step a) is negatively correlated with the humoral immune response of said subject to said vaccine.

2. The in vitro method according to claim 1 comprising the step of comparing said level of one or more gene expression to a control reference value wherein:

- a low level of one or more gene expression compared to said control reference value is predictive that said subject is a high risk to be a “high responder” to said vaccine.

3. The in vitro method according to any one of claim 1 to 2, wherein the sample is a blood sample.

4. The in vitro method according to any one of claim 1 or 3, wherein the level of one or more gene expression is determined at mRNA level.

5. The in vitro method according to any one of claim 1 or 4, wherein the level of the gene expression is determined with one, two, three, four gene selected from the group consisting of IGLV8-61, BLK, EBF1 and IFNal6 gene.

6. The in vitro method according to any one of claim 1 or 5, wherein the vaccine contain an antigen selected from the group consisting of attenuated and inactivated viral and bacterial pathogens, purified macromolecules, polysaccharides, toxoids, recombinant antigens, organisms containing a foreign gene from a pathogen, synthetic peptides, polynucleic acids (DNA, RNA), antibodies and tumor cells.

7. The in vitro method according to any one of claim 1 or 6, comprising an additional step consisting of measuring in a sample obtained from said subject the level Eubacterium in stool sample and Prevotella in skin sample.

8. The in vitro method according to claim 7, wherein the level Eubacterium in stool sample and Prevotella in skin sample is performed by using 16S ribosomal sequencing.

9. A kit, comprising:

- at least a mean for determining one or more gene expression level selected from a group of gene consisting of: IGLV8-61, BLK, EBF1 and IFNal6 gene and

- instructions for use, for use to assess a subject’s humoral immune response to a vaccine.

10. A kit for use according to claim 9, comprising:

- amplification primers and/or probe for determining one or more gene expression level selected from a group of gene consisting of: IFNal6, IGLV8-61, BLK, and EBF 1 gene,

- instructions for use.

11. Method for vaccinating a subject in need thereof with an antigen wherein, prior to vaccination, the level of one or more gene expression level selected from a group of gene consisting of: IFNal6IGLV8-61, BLK and EBF1 gene obtained from said subject, have been detected by one of the method of claim 1 to 8.