Implications For Vaccine Design Research Articles

Evasion of Host Immunity by Virulent Salmonella: Implications for Vaccine Design

Dendritic cells (DCs) are professional antigen presenting cells (APCs) capable of linking innate and adaptive immunity during infection. After recognition of pathogen-associated molecular patterns (PAMPs), DCs can engulf, process and present bacteria-derived antigens on MHC molecules to T cells. Because of the key role that DCs play on the initiation of innate and adaptive immunity, alterations in their function could render the host susceptible to bacterial dissemination. Consistent with this notion, is the observation that several pathogenic bacteria have evolved mechanisms to impair the DC capacity to prime naïve T cells. One of such bacteria is Salmonella enterica serovar Typhimurium, which causes a typhoid-like disease in mice and gastroenteritis in humans. Recent studies have shown that virulent Salmonella can use intestinal DCs to spread inside the host, evading T cell priming. The avoidance of T cell recognition by Salmonella is in large part achieved by the activity of gene products encoded on Salmonella Pathogenicity Islands -1 and - 2. The understanding of some of the remarkable molecular virulence mechanisms displayed by Salmonella has contributed to the design of new vaccines capable of inducing protective immunity against this pathogen in mouse models. Here we describe recent data underscoring the virulence mechanisms used by Salmonella to exploit DC function and discuss strategies based on this new knowledge aimed at the design of new efficient and safe vaccines against this pathogen.

A mechanism for glycoconjugate vaccine activation of the adaptive immune system and its implications for vaccine design

Although glycoconjugate vaccines have provided enormous health benefits globally, they have been less successful in significant high-risk populations. Exploring novel approaches to the enhancement of glycoconjugate effectiveness, we investigated molecular and cellular mechanisms governing the immune response to a prototypical glycoconjugate vaccine. In antigen-presenting cells, a carbohydrate epitope is generated upon endolysosomal processing of group B streptococcal type III polysaccharide coupled to a carrier protein. In conjunction with a carrier protein-derived peptide, this carbohydrate epitope binds to major histocompatibility class II (MHCII) and stimulates carbohydrate-specific CD4+ T-cell clones to produce interleukins 2 and 4—cytokines essential for providing T-cell help to antibody-producing B cells. An archetypical glycoconjugate vaccine constructed to maximize the presentation of carbohydrate epitopes recognized by T cells is 50–100 times more potent and significantly more protective in an animal model of infection than is a currently used vaccine construct.

Loss of T Cell Antigen Recognition Arising from Changes in Peptide and Major Histocompatibility Complex Protein Flexibility: IMPLICATIONS FOR VACCINE DESIGN

Modification of the primary anchor positions of antigenic peptides to improve binding to major histocompatibility complex (MHC) proteins is a commonly used strategy for engineering peptide-based vaccine candidates. However, such peptide modifications do not always improve antigenicity, complicating efforts to design effective vaccines for cancer and infectious disease. Here we investigated the MART-1(27-35) tumor antigen, for which anchor modification (replacement of the position two alanine with leucine) dramatically reduces or ablates antigenicity with a wide range of T cell clones despite significantly improving peptide binding to MHC. We found that anchor modification in the MART-1(27-35) antigen enhances the flexibility of both the peptide and the HLA-A*0201 molecule. Although the resulting entropic effects contribute to the improved binding of the peptide to MHC, they also negatively impact T cell receptor binding to the peptide·MHC complex. These results help explain how the "anchor-fixing" strategy fails to improve antigenicity in this case, and more generally, may be relevant for understanding the high specificity characteristic of the T cell repertoire. In addition to impacting vaccine design, modulation of peptide and MHC flexibility through changes to antigenic peptides may present an evolutionary strategy for the escape of pathogens from immune destruction.

Insights into HLA-Restricted T Cell Responses in a Novel Mouse Model of Dengue Virus Infection Point toward New Implications for Vaccine Design

The frequency of dengue virus (DENV) infection has increased dramatically in the last few decades, and the lack of a vaccine has led to significant morbidity and mortality worldwide. To date, a convenient murine system to study human T cell responses to DENV has not been available. Mice transgenic for HLA are widely used to model human immune responses, and it has been shown that mouse-passaged DENV is able to replicate to significant levels in IFN-α/βR(-/-) mice. To cover a wide range of HLA phenotypes, we backcrossed IFN-α/βR(-/-) mice with HLA A*0201, A*0101, A*1101, B*0702, and DRB1*0101-transgenic mice. A DENV proteome-wide screen identified a total of 42 epitopes across all HLA-transgenic IFN-α/βR(-/-) strains tested. In contrast, only eight of these elicited responses in the corresponding IFN-α/βR(+/+) mice. We were able to identify T cell epitopes from 9 out of the 10 DENV proteins. However, the majority of responses were derived from the highly conserved nonstructural proteins NS3 and NS5. The relevance of this model is further demonstrated by the fact that most of the epitopes identified in our murine system are also recognized by PBMC from DENV-exposed human donors, and a dominance of HLA B*0702-restricted responses has been detected in both systems. Our results provide new insights into HLA-restricted T cell responses against DENV, and we describe in this study a novel murine model that allows the investigation of T cell-mediated immune mechanisms relevant to vaccine design.

Human papillomaviruses 53 and 66: Clinical aspects and genetic analysis

Variants of HPV types may have different oncogenic potential. While HPV 16 and 18 variants have been extensively studied, little is known on the less frequent high-risk types such as HPV 53 and 66. Here, we analyzed the genetic variability of HPV 53 and 66 by sequencing the E6, E7, L1 genes and the Long Control Region (LCR) sequences of HPV 53 and HPV 66 from infected women. Fisher's exact-test was performed to correlate viral variants with cervical lesions. Higher-order interactions among identified mutations were analyzed by co-variation and cluster analyses. Antigenic-index alterations following L1 mutations were predicted by Jameson–Wolf algorithm.In HPV53, novel variants were identified in L1 (N=9) and E6 (N=1) genes. The novel L1 mutation P432L was statistically associated with L-SIL lesions (P=0.04) and its development reduced the L1 predicted antigenicity (up to −2.3 for Glu433). HPV 53 E6 and L1 sequences clustered phylogenetically into two main clades.In HPV 66, novel polymorphisms were identified in L1 (N=4) and E6 (N=4) genes. The L1 protein mutations S405P and D458N were exclusively found in patients with L-SILs. Seven E7 variants and 10 LCR variants were for the first time analyzed.Novel HPV 53 and 66 variants were identified in this study. Some of these mutations were significantly associated with L-SIL lesions and affected the antigenic index of the L1 protein with possible interesting implications in vaccine design.

Promotion of the neutralization-competent structure of the HIV-1 gp41 membrane proximal external region when tethered to its native transmembrane domain, and expressed in the context of the plasma membrane: implications for vaccine design (53.24)

Abstract The limited success of vaccines targeting the MPER, a target of three broadly neutralizing (bNt) monoclonal antibodies (MAbs), we hypothesize, reflects the difficulty of mimicking the neutralization-competent structure (NCS) of the MPER. We determined the contribution of the amino-acid sequence and the transmembrane domain (TM), to the antigenicity of the MPER in the context of the plasma membrane. DNA constructs encoding various gp41 ectodomain fragments, and the TM of either the platelet-derived growth factor receptor (PGDFR), or that of gp41, were produced and transiently expressed in COS-7 cells. Constructs expressing the MPER tethered to the gp41 TM followed by a 27-residue cytoplasmic tail fragment, MPER-TM1, produced optimal binding of MAbs 2F5, 4E10 and Z13e1. A series of 24 single amino-acid substitutions in the MPER-TM1 revealed critical binding residues for the three MAbs; similar substitutions were previously shown to ablate Ab-mediated viral neutralization. Neutralization-incompetent 2F5 Fab and 4E10 IgG mutant Abs failed to bind MPER-TM1, yet retained the ability to bind to peptide epitopes, indicating the plasma-membrane expressed MPER-TM1 closely approaches the NCS of the MPER. Substitution of the TM of gp41 with that from the PGDFR reduced binding by MAb 4E10, but not MAbs 2F5 or Z13e1. Our studies reveal that the gp41 TM appears to play a pivotal role both in orienting the 4E10 epitope, and affecting exposure of the MPER epitopes for all three bNtMAbs.

Investigating the role of 4-1BB and 4-1BBL in dendritic cell immunobiology (113.6)

Abstract The tumour necrosis factor (TNF) receptor family member 4-1BB, and its TNF family ligand, 4-1BBL, have been thoroughly investigated as potent modulators of the CD8 T cell response. During a viral infection, 4-1BBL on APC, binding to 4-1BB on activated T cells mediates survival signaling in activated and memory CD8 T cells. Similarly to T cells, 4-1BB is also induced on dendritic cells (DC) following activation; however, how 4-1BB and 4-1BBL modulate DC biology is poorly understood. We provide evidence that DC express both 4-1BB and 4-1BBL upon activation with a microbial stimulus in vitro, and that these molecules interact on maturing DC. Using an in vivo DC vaccination model, we demonstrate that in the absence of 4-1BBL on the DC, there is a 2-3 fold defect in the ability of these DC to generate influenza-specific CD8 T cells in a WT host. This defect is not observed when 4-1BB-/- hosts are vaccinated with WT DC, suggesting that 4-1BBL on the DC is not acting through 4-1BB in the host, but intrinsically on the DC. Even more striking is that 4-1BB-/- DC do not show this defect in the generation of flu-specific CD8 T cells, raising the possibility of a 4-1BB-independent 4-1BBL mechanism. In that regard, we further show that anti-4-1BBL antibodies can induce Erk activation in WT DC, but not in 4-1BBL-/- DC. These results identify 4-1BBL as a DC modulator and have overall implications for vaccine design.

Specific regions of the ricin toxin A subunit are involved in eliciting protective antibodies: implications for vaccine design (53.5)

Abstract Efforts to develop an effective vaccine against ricin are focused on engineering attenuated and stable recombinant forms of the toxin’s enzymatic A subunit (RTA). While several candidate antigens are in development, vaccine design and efficacy studies are being undertaken in the absence of a fundamental understanding of the regions of RTA that are critical in eliciting protective immunity. To address this issue, we recently identified six distinct immunodominant linear regions on RTA. Using monoclonal antibodies (MAbs) specific to each region, we determined that protective antibodies were directed against α-helices located in RTA folding domains 1 and 2, whereas non-neutralizing antibodies recognized random coils and loops that were primarily confined to folding domain 3. This suggests that neutralizing and non-neutralizing antibodies target distinct domains on RTA. In support of this hypothesis, we now report that the vast majority of conformation-dependent ricin neutralizing MAbs bind to RTA 1-33/44-198, a recombinant RTA deletion mutant lacking folding domain 3. Furthermore, preliminary studies in mice suggest that RTA 1-33/44-198 is more effective than an attenuated RTA point mutant at eliciting neutralizing antibodies. These data offer insights into the immunodominant and structural determinants on RTA that give rise to protective immunity, and for the first time provide an immunological rationale for ricin vaccine design.

85 IL28B GENETIC STATUS IN ASSOCIATION WITH HCV SPECIFIC T-CELL IMMUNITY

Introduction: Hepatitis E virus (HEV) infection usually results in an acute, self-limiting hepatitis in immunocompetent individuals while it may rarely progress to fulminant hepatic failure. Several cases of chronic evolution of HEV infections have been described recently in French, Dutch and German immunosuppressed patients. However immune correlates of clearance or persistence of HEV have not been described so far. Aims of this study were to i. investigate whether HEV persistence is associated with impaired immune responses and ii. identify potential epitopic regions in HEV genome that may elicit strong immune cellular responses to be considered for a potential vaccine. Methods: Immune responses against HEV were studied in a total of 29 subjects including 21 healthy immunocompetent individuals (anti-HEV-positive; n = 13 (“exposed”); anti-HEV-negative n =8 (“no exposure”)) and 8 anti-HEV-positive patients after organ transplantation. 4 transplanted patients evolved to chronic hepatitis while 4 subjects were followed and cleared HEV spontaneously. PBMCs were stimulated with HEV overlapping peptide pools (spanning ORF2 and ORF3) or individual peptides. T-cell proliferation was measured after 7 days by CFSE (5, 6-carboxyfluorescein diacetate succinimidyl ester) assay. Cytokine secretion was investigated by intra-cellular cytokine staining (IFN-gamma, MIP-1beta and TNF-alpha). Results: Multispecific (positive responses for more at least 2 peptide pools) and strong HEV-specific CD4/CD8 T-cell were detectable in 77%/62% of HEV-seropositive healthy subjects respectively. No HEV-specific responses were observed in any of the HEV-unexposed individuals. In transplant recipients, multispecific but moderate T-cell responses were detectable in resolved individuals while chronic patients did not show any specific T cell response. Using individual peptides, immune responses were narrowed-down to two epitopes that presented very strong CD8 responses. These results were further corroborated by cytokine production, where IFN-gamma and MIP-1beta secretion was observed only in seropositive healthy individuals. Conclusion: These results suggest that chronic HEV infection is associated with impaired HEV-specific adaptive immune responses. Identification of immunodominant epitopes may have implications for vaccine design. 85 IL28B GENETIC STATUS IN ASSOCIATION WITH HCV SPECIFIC T-CELL IMMUNITY C. Bucci, M. Rahman, I. Humphreys, A.C. Brown, M. Lucas, K. Pfafferott, J. Collier, D. O’Donnell, E. Stafford, P. Klenerman, S. Gaudieri, E. Barnes. Department of Hepatology, Nuffield Department of Medicine & Oxford NIHR BRC, Nuffield Department of Medicine, University of Oxford, Oxford, UK; Institute of Immunology and Infectious Diseases, Murdoch University, Perth, WA, Australia; Hepatology & Oxford NIHR BRC, University of Oxford, Oxford, UK; School of Anatomy and Human Biology and Centre for Forensic Science, University of Western Australia, Perth, WA, Australia E-mail: cristinabucci@hotmail.it

The vitamin D receptor and inducible nitric oxide synthase associated pathways in acquired resistance to Cooperia oncophora infection in cattle

Cooperia oncophora is an economically important gastrointestinal nematode in ruminants. Acquired resistance to Cooperia oncophora infection in cattle develops rapidly as a result of prior infections. Naïve cattle, when given a primary infection of high-dose infective L3 larvae, develop a strong immunity to subsequent reinfection. Compared to primary infection, reinfection resulted in a marked reduction in worm establishment. In order to understand molecular mechanisms underlying the development of acquired resistance, we characterized the transcriptomic responses of the bovine small intestine to a primary infection and reinfection. A total of 23 pathways were significantly impacted during infection. The vitamin D receptor activation was strongly induced only during reinfection, suggesting that this pathway may play an important role in the development of acquired resistance via its potential roles in immune regulation and intestinal mucosal integrity maintenance. The expression of inducible nitric oxide synthase (NOS2) was strongly induced during reinfection but not during primary infection. As a result, several canonical pathways associated with NOS2 were impacted. The genes involved in eicosanoid synthesis, including prostaglandin synthase 2 (PTGS2 or COX2), remained largely unchanged during infection. The rapid development of acquired resistance may help explain the lack of relative pathogenicity by Cooperia oncophora infection in cattle. Our findings facilitate the understanding of molecular mechanisms underlying the development of acquired resistance, which could have an important implication in vaccine design.

Insights into HLA-Restricted T Cell Responses in a Novel Mouse Model of Dengue Virus Infection Point toward New Implications for Vaccine Design

Insights into HLA-Restricted T Cell Responses in a Novel Mouse Model of Dengue Virus Infection Point toward New Implications for Vaccine Design

Antigen-bearing dendritic cells regulate the diverse pattern of memory CD8 T-cell development in different tissues

Memory T cells of the effector type (T(EM)) account for the characteristic rapidity of memory T-cell responses, whereas memory T cells of the central type (T(CM)) account for long-lasting, vigorously proliferating memory T-cell responses. How antigen-stimulated (primed) T cells develop into different memory T-cell subsets with diverse tissue distributions is largely unknown. Here we show that after respiratory tract infection of mice with influenza virus, viral antigen associated with dendritic cells (DCs) was abundant in lung-draining lymph nodes (DLN) and the spleen for more than a week but was scant and transient in nondraining lymph nodes (NDLN). Correspondingly, activated CD8 T cells proliferated extensively in DLN and the spleen but minimally in NDLN. Strikingly, however, although most persisting CD8 T cells in DLN and spleen exhibited the T(EM) phenotype, those persisting in NDLN exhibited the T(CM) phenotype. Reducing antigen exposure by depleting DCs at the peak of primary T-cell responses enhanced the development of T(CM), whereas subjecting primed CD8 T cells from NDLN to additional antigen stimulation inhibited T(CM) development. These findings demonstrate that differences in persistence of antigen-bearing DCs in various tissues regulate the tissue-specific pattern of memory CD8 T-cell development. The findings have significant implications for design of vaccines and immunization strategies.

HIV-1 Superinfection and its Implications for Vaccine Design

HIV-1 superinfection, which refers to a subsequent HIV-1 infection from a different source partner after the first HIV-1 infection is established, has now been well documented in multiple populations. Some studies suggest that the risk of superinfection may be close to that of initial infection, suggesting that the immunity induced by chronic HIV-1 infection may not be adequate to confer protection from another HIV-1 strain. Detailed studies that examined immune responses in individuals who became superinfected generally support this hypothesis, but such studies have been limited. Indeed, superinfection represents one of the few settings, apart from vaccine trials, where there is an opportunity to gain insights into the role of HIV-specific immunity in protection in humans, and this should be exploited. Likewise, studies of superinfection in HIV-1 positive individuals on antiretroviral therapy who continue to be exposed to HIV could provide insight into the role of antiretroviral treatment in protecting from HIV-1 infection, a concept that is also being explored for its potential to prevent a first HIV-1 infection. To address these questions, larger population-based studies that define the incidence and timing of superinfection and include collection of samples for immunological studies are needed.

Molecular characterization of equine rotaviruses circulating in Argentinean foals during a 17-year surveillance period (1992–2008)

P[12]G3 and P[12]G14 equine rotaviruses (ERVs) are epidemiologically important in horses. In Argentina, the prevalent ERV strains have been historically P[12]G3. The aim of this study was the detection and characterization of ERV strains circulating in foals in Argentina during a 17-year study (1992–2008). Additionally, the gene sequences of VP7, VP4 and NSP4 encoding genes of representative Argentinean ERV strains were determined and phylogenetic analyses were performed to elucidate the evolutionary relationships of the ERV strains in Argentina. ERVs were detected in 165 (21%) out of 771 diarrheic stool samples, which corresponded to 45 (39%) of 116 outbreaks from the surveyed thoroughbred horse farms. From the positive cases, 51% ( n = 23) were G3, 33% ( n = 15) were G14, 4% ( n = 2) represented a G3 + G14 mixed infection and 11% ( n = 5) of the cases could not be characterized. G3 ERV was detected during the entire period, while G14 ERV was first detected in 2000 and increased its incidence specially in 2006 and 2007. All the analyzed strains belonged to the VP4 P[12] genotype, except for one G3 case which belonged to the P[3] genotype, constituting the first report of a P[3]G3 ERV strain. Phylogenetic analysis of VP7 protein revealed that the G3 Argentinean ERV strains clustered with ERVs from Ireland, while the G14 Argentinean ERV strains formed a distinct cluster within the G14 genotype. The VP4 of the P[12] ERV strains clustered with P[12] strains from Ireland and France. The NSP4 of the Argentinean ERV strains clustered with the NSP4 genotype E12, along with those of guanaco and bovine strains from Argentina, suggesting the a close evolutionary relationship among these Argentinean strains. The results of this study showed changes in the incidence of G3 and G14 during the studied period. The increase in the frequency of G14 ERV, not included in the vaccine, in the second half of the period, may have implications for vaccine design.

A peptide mimotope of hepatitis C virus E2 protein is immunogenic in mice and block human anti‐HCV sera

Conformational B-cell epitopes on the HCV E2 protein recognized by human antibodies were characterized by the use of a peptide mimotope named K1. K1 was identified by two HCV anti-E2 monoclonal antibodies (mAbs) following selection and purification of phage clones containing a 15-mer random peptide insert. Murine antisera to the mimotope K1 recognized the E2 protein. Five of eight human sera from patients who had cleared HCV recognized the K1 mimotope. Binding to E2 in four individuals with the capacity to block E2-CD81 interaction was inhibited by the mimotope K1. The results demonstrate that anti-E2 antibodies in sera from patients who have cleared HCV infection are directed against a conformational B-cell epitope on E2 that can be mimicked with linear synthetic peptides. These findings could have implications for vaccine design by employing linear mimotopes to direct B-cell responses against those specific E2 epitopes that may correlate with immunity.

Adaptation of HIV-1 envelope gp120 to humoral immunity at a population level

By comparing HIV-1 variants from people who became infected at the beginning of the epidemic and from people who have recently contracted the virus, we observed an enhanced resistance of the virus to antibody neutralization over time, accompanied by an increase in the length of the variable loops and in the number of potential N-linked glycosylation sites on the HIV-1 envelope gp120 subunit. The enhanced neutralization resistance of HIV-1 in contemporary seroconverters coincided with the poorer elicitation of neutralizing antibody responses, which may have implications for vaccine design.

The Achilles heel of BCG

There have been multiple explanations put forward to try to explain the variable efficacy of the BCG vaccine. Here I propose the new hypothesis that the primary flaw of BCG is its inability to effectively establish a population of central memory T cells. Instead, the vaccine establishes immunity represented by effector memory T cells; these distribute in the lungs and may protect humans for 10-15 years but are gradually lost. With no central memory response to compensate, the individual loses any further resistance to tuberculosis. This may have serious implications for vaccine design, given the emphasis on developing recombinant forms of BCG.

Ratio of HCV structural antigens in protein-based vaccine formulations is critical for functional immune response induction

HCV (hepatitis C virus) infection is among the leading causes of chronic liver disease, but currently there is no vaccine available. Data have accumulated about the importance of targeting different HCV antigens in vaccine candidate preparations. Here, a surface response study to select the optimal ratio of recombinant HCV structural antigens in a vaccine preparation, capable of generating in vivo functional cellular immune response in mice, was performed. The immunogenicity of the selected HCV structural protein mixture (Co-E1-E2) in mice and African green monkeys, after five doses of immunization, was also demonstrated. Specific T-cell proliferative response against HCV structural antigens was induced in vaccinated mice. Moreover, on challenge with recombinant HCV VV (vaccinia virus), all mice controlled the viraemia and 80% were protected. On the other hand, monkeys immunized with Co-E1-E2 developed antibodies, specifically directed to region 412-438 of E2 protein, that include an epitope implicated in HCV neutralization, in addition to a specific proliferative response against HCV Core and E2 proteins. These results indicated that the optimal amount and ratio of HCV recombinant proteins should be taken into account to elicit a successful immune response against HCV and therefore have important implications for vaccine design.

The Evolution of HIV-1 Diversity in Rural Cameroon and its Implications in Vaccine Design and Trials

West-Central Africa is an epicenter of the HIV pandemic; endemic to Cameroon are HIV-1 viruses belonging to all (sub)subtypes and numerous Circulating Recombinant Forms (CRFs). The rural villages of Cameroon harbor many strains of HIV-1, though these areas are not as well monitored as the urban centers. In the present study, 82 specimens obtained in 2000 and 2001 from subjects living in the rural villages of the South and West Regions of Cameroon were subtyped in gag, pol, and env and compared to 90 specimens obtained in 2006–2008 in the same regions, in order to analyze HIV-1 evolution in these rural areas. It was found that in the South Region, the proportion of unique recombinant forms (URFs) remained constant (∼40%), while the amount of URFs containing fragments of a CRF increased by 25%. (Sub)subtypes A1, F2, H, and K, and CRF09_cpx, identified in 2000 and 2001, were replaced by CRFs 01_AE, 13_cpx, 14_BG, and 18_cpx in 2006–2008. In the West Region, (sub)subtypes A2, C, G, and H, and CRFs 01_AE and 09_cpx, identified in 2000–2001, were replaced by sub-subtype A1 and CRFs 25_cpx and 37_cpx in 2006–2008. The proportion of URFs in the West Region dropped significantly over the time period by 43%. In both Regions, the proportion of CRF02_AG increased at all loci. These findings demonstrate that the evolution of HIV-1 is distinct for each endemic region, and suggests that the proportion of URFs containing CRF fragments is increasing as the genetic identity of the virus continues to shift dramatically. This highlights the concern that subtype-specific vaccines may not be relevant in Cameroon, and that the distribution of viral diversity in these regions of Cameroon must be carefully monitored.

Genetic variation in Staphylococcus aureus surface and immune evasion genes is lineage associated: implications for vaccine design and host-pathogen interactions

BackgroundS. aureus is a coloniser and pathogen of humans and mammals. Whole genome sequences of 58 strains of S. aureus in the public domain and data from multi-strain microarrays were compared to assess variation in the sequence of proteins known or putatively interacting with host.ResultsThese included 24 surface proteins implicated in adhesion (ClfA, ClfB, Cna, Eap, Ebh, EbpS, FnBPA, FnBPB, IsaB, IsdA, IsdB, IsdH, SasB, SasC, SasD, SasF, SasG, SasH, SasK, SdrC, SdrD, SdrE, Spa and SraP) and 13 secreted proteins implicated in immune response evasion (Coa, Ecb, Efb, Emp, EsaC, EsxA, EssC, FLIPr, FLIPr like, Sbi, SCIN-B, SCIN-C, VWbp) located on the stable core genome. Many surface protein genes were missing or truncated, unlike immune evasion genes, and several distinct variants were identified. Domain variants were lineage specific. Unrelated lineages often possess the same sequence variant domains proving that horizontal transfer and recombination has contributed to their evolution. Surprisingly, sequenced strains from four animal S. aureus strains had surface and immune evasion proteins remarkably similar to those found in human strains, yet putative targets of these proteins vary substantially between different hosts. This suggests these proteins are not essential for virulence. However, the most variant protein domains were the putative functional regions and there is biological evidence that variants can be functional, arguing they do play a role.ConclusionSurface and immune evasion genes are candidates for S. aureus vaccines, and their distribution and functionality is key. Vaccines should contain cocktails of antigens representing all variants or they will not protect against naturally occurring S. aureus populations.