Epitope-specific Responses Research Articles

Modulation of epitope-specific antibody response by HIV Env immune-complex vaccines

Abstract HIV-1 envelope (Env) antigen is an important target for both neutralizing and non-neutralizing antibodies (Ab) against the virus. In the RV144 vaccine trial, correlate for reduced risk of HIV acquisition is the presence of binding but non-neutralizing Ab to V1V2 and V3 loops of HIV Env gp120. However, induction of high titer, durable, and cross-reactive Ab responses against these Env regions by vaccination is still an elusive goal. Our past studies have shown that V3 immunogenicity can be significantly enhanced by vaccination with gp120/mAb immune complexes (IC) vs gp120 alone. Current study further examined parameters that confer enhanced immunogenicity to IC by testing in mice IC vaccines made with different HIV Env gp120 (clade B vs clade E) and mAb of different specificities (V3, V2, or CD4 binding site (CD4bs)). In addition, IC made of clade C gp140 and CD4bs mAb were also tested. The data showed that Greater titers of cross-reactive V3-binding Ab were induced by IC made of clade B gp120 and mAb to V2 or CD4bs as compare with gp120 alone, although the gp120/CD4bs IC was more potent in eliciting neutralizing V3 Ab. IC made with clade E gp120 or clade C gp140 did not show the same effects.Greater Ab responses to V1V2 were induced by IC made of clade E gp120 vs gp120 alone, regardless of the mAb used for IC, but the Ab were not neutralizing. IC made with clade B gp120 and clade C gp140 did not enhance V1V2 Ab titers. These results demonstrate the capacity of Env/mAb IC vaccines to modulate induction of Ab responses to V1V2 and V3, and this activity is dependent on both Env proteins and mAb used to form IC. Further exploration using a cocktail of IC is proposed to improve Ab repertoires generated upon vaccination.

Abstract 395: Evaluation of Antibody Responses in Mice Toward Apolipoprotein A-I

Antibodies targeting apolipoprotein A-I (ApoA-I) have been documented in patients suffering from chronic inflammation associated with obesity, autoimmunity and cardiovascular disease (CVD). Anti-ApoA-I antibodies are thought to be serum markers for CVD and not responsible for disease progression, but their exact role is poorly understood. We hypothesize that the antibody response to ApoA-I is highly nuanced with both protective and pathologic antibodies targeting modified or unmodified epitopes. We sought to induce ApoA-I specific autoantibodies in mice targeting specific modified and unmodified epitopes to study their nuanced role in disease progression. To achieve this goal we immunized mice with an immunogenic liposomal formulation containing modified or unmodified peptides derived from ApoA-I. The epitope-specific antibody response toward each epitope and the full length protein were quantified by ELISA. Mice immunized with formulations containing peptide were compared to control mice receiving either peptide or adjuvant alone. Furthermore, antibody responses toward ApoA-I from mice fed normal chow were compared to mice fed a western diet. Our vaccine strategy induced a robust immune response toward the peptide epitopes along with the full length protein. The oxidized peptide also appeared to be more immunogenic than the unmodified, but the mechanism of this observation is unclear. Antibody responses toward ApoA-I were observed after immunization with adjuvant alone suggesting that acute inflammation induces a response to ApoA-I. Studies are currently underway to evaluate the affinity of the anti-ApoA-I antibodies for HDL along with their role in cholesterol efflux and atherosclerosis progression. Correlating these findings with studies in human patients will generate insight into the nuanced antibody responses toward ApoA-I in CVD.

Differential Antibody Responses to Conserved HIV-1 Neutralizing Epitopes in the Context of Multivalent Scaffolds and Native-Like gp140 Trimers.

ABSTRACTBroadly neutralizing antibodies (bNAbs) have provided valuable insights into the humoral immune response to HIV-1. While rationally designed epitope scaffolds and well-folded gp140 trimers have been proposed as vaccine antigens, a comparative understanding of their antibody responses has not yet been established. In this study, we probed antibody responses to the N332 supersite and the membrane-proximal external region (MPER) in the context of heterologous protein scaffolds and native-like gp140 trimers. Ferritin nanoparticles and fragment crystallizable (Fc) regions were utilized as multivalent carriers to display scaffold antigens with grafted N332 and MPER epitopes, respectively. Trimeric scaffolds were also identified to stabilize the MPER-containing BG505 gp140.681 trimer in a native-like conformation. Following structural and antigenic evaluation, a subset of scaffold and trimer antigens was selected for immunization in BALB/c mice. Serum binding revealed distinct patterns of antibody responses to these two bNAb targets presented in different structural contexts. For example, the N332 nanoparticles elicited glycan epitope-specific antibody responses that could also recognize the native trimer, while a scaffolded BG505 gp140.681 trimer generated a stronger and more rapid antibody response to the trimer apex than its parent gp140.664 trimer. Furthermore, next-generation sequencing (NGS) of mouse splenic B cells revealed expansion of antibody lineages with long heavy-chain complementarity-determining region 3 (HCDR3) loops upon activation by MPER scaffolds, in contrast to the steady repertoires primed by N332 nanoparticles and a soluble gp140.664 trimer. These findings will facilitate the future development of a coherent vaccination strategy that combines both epitope-focused and trimer-based approaches.

CD4 T cell epitope specificity determines follicular versus non-follicular helper differentiation in the polyclonal response to influenza infection or vaccination.

Follicular helper T cells (Tfh) are essential for B cell production of high-affinity, class-switched antibodies. Much interest in Tfh development focuses on the priming environment of CD4 T cells. Here we explored the role that peptide specificity plays in the partitioning of the polyclonal CD4 T cell repertoire between Tfh and NonTfh lineages during the response to influenza. Surprisingly, we found that CD4 T cells specific for different epitopes exhibited distinct tendencies to segregate into Tfh or NonTfh. To alter the microenvironment and abundance, viral antigens were introduced as purified recombinant proteins in adjuvant as native proteins. Also, the most prototypical epitopes were expressed in a completely foreign protein. In many cases, the epitope-specific response patterns of Tfh vs. NonTfh persisted. The functional TcR avidity of only a subset of epitope-specific cells correlated with the tendency to drive a Tfh response. Thus, we conclude that in a polyclonal CD4 T cell repertoire, features of TcR-peptide:MHC class II complex have a strong deterministic influence on the ability of CD4 T cells to become a Tfh or a NonTfh. Our data is most consistent with at least 2 checkpoints of Tfh selection that include both TcR affinity and B cell presentation.

Immunogenicity of multi-epitope-based vaccine candidates administered with the adjuvant Gp96 against rabies.

Rabies, a zoonotic disease, causes > 55,000 human deaths globally and results in at least 500 million dollars in losses every year. The currently available rabies vaccines are mainly inactivated and attenuated vaccines, which have been linked with clinical diseases in animals. Thus, a rabies vaccine with high safety and efficacy is urgently needed. Peptide vaccines are known for their low cost, simple production procedures and high safety. Therefore, in this study, we examined the efficacy of multi-epitope-based vaccine candidates against rabies virus. The ability of various peptides to induce epitope-specific responses was examined, and the two peptides that possessed the highest antigenicity and conservation, i.e., AR16 and hPAB, were coated with adjuvant canine-Gp96 and used to prepare vaccines. The peptides were prepared as an emulsion of oil in water (O/W) to create three batches of bivalent vaccine products. The vaccine candidates possessed high safety. Virus neutralizing antibodies were detected on the day 14 after the first immunization in mice and beagles, reaching 5-6 IU/mL in mice and 7-9 IU/mL in beagles by day 28. The protective efficacy of the vaccine candidates was about 70%-80% in mice challenged by a virulent strain of rabies virus. Thus, a novel multi-epitope-based rabies vaccine with Gp96 as an adjuvant was developed and validated in mice and dogs. Our results suggest that synthetic peptides hold promise for the development of novel vaccines against rabies.

Functional Transplant of a Dengue Virus Serotype 3 (DENV3)-Specific Human Monoclonal Antibody Epitope into DENV1.

The four dengue virus (DENV) serotypes, DENV1 through 4, are endemic throughout tropical and subtropical regions of the world. While first infection confers long-term protective immunity against viruses of the infecting serotype, a second infection with virus of a different serotype carries a greater risk of severe dengue disease, including dengue hemorrhagic fever and dengue shock syndrome. Recent studies demonstrate that humans exposed to DENV infections develop neutralizing antibodies that bind to quaternary epitopes formed by the viral envelope (E) protein dimers or higher-order assemblies required for the formation of the icosahedral viral envelope. Here we show that the quaternary epitope target of the human DENV3-specific neutralizing monoclonal antibody (MAb) 5J7 can be partially transplanted into a DENV1 strain by changing the core residues of the epitope contained within a single monomeric E molecule. MAb 5J7 neutralized the recombinant DENV1/3 strain in cell culture and was protective in a mouse model of infection with the DENV1/3 strain. However, the 5J7 epitope was only partially recreated by transplantation of the core residues because MAb 5J7 bound and neutralized wild-type (WT) DENV3 better than the DENV1/3 recombinant. Our studies demonstrate that it is possible to transplant a large number of discontinuous residues between DENV serotypes and partially recreate a complex antibody epitope, while retaining virus viability. Further refinement of this approach may lead to new tools for measuring epitope-specific antibody responses and new vaccine platforms. Dengue virus is the most important mosquito-borne pathogen of humans worldwide, with approximately one-half the world's population living in regions where dengue is endemic. Dengue immunity following infection is robust and thought to be conferred by antibodies raised against the infecting virus. However, the specific viral components that these antibodies recognize and how they neutralize the virus have been incompletely described. Here we map a region on dengue virus serotype 3 recognized by the human neutralizing antibody 5J7 and then test the functional significance of this region by transplanting it into a serotype 1 virus. Our studies demonstrate a region on dengue virus necessary for 5J7 binding and neutralization. Our work also demonstrates the technical feasibility of engineering dengue viruses to display targets of protective antibodies. This technology can be used to develop new dengue vaccines and diagnostic assays.

Synthesis and immunological evaluation of peptide-based vaccine candidates against malaria

Background: Malaria, caused by the protozoan parasite Plasmodium, is one of the main causes of morbidity and mortality of the whole human population.Intensive, ongoing research aims to develop an effective vaccine against malaria; however, it has been unsuccessful for over a century. The circumsporozoite protein (CSP) plays crucial a role in the parasite life cycle. CSP is the most dominant surface antigen of the initial pre-erythrocytic stage. We designed vaccine constructs using four different CD4+ and CD8+ T cell epitopes derived from the CSP and used the lipid core peptide (LCP) as a self-adjuvanting delivery system. Methods: All the constructs were synthesized using microwave-assisted solid phase peptide synthesis (SPPS). Immunological evaluation was carried out following subcutaneous administration of LCP-based vaccine candidates in a BALB/c mouse model. Interferon gamma (IFN-γ) production was used to measure the induction of epitope-specific cellular immune responses after vaccination. Results: Self-adjuvanting LCP malaria vaccines composed of different epitopes were synthesized.To determine whether the vaccine candidates were able to induce cellular immunity, mice were immunized with LCP constructs or peptide epitopes adjuvanted with cholera toxin.Two of the tested constructs induced a high level of INF-γ in mice after subcutaneous immunization. Conclusions: We have demonstrated here for the first time that the LCP delivery system induced epitopespecific cellular immune responses against an antigen derived from Plasmodium.

Immunoreactivity of chimeric proteins carrying poliovirus epitopes on the VP6 of rotavirus as a vector

Rotavirus and poliovirus continue to present significant risks and burden of disease to children in developing countries. Developing a combined vaccine may effectively prevent both illnesses and may be advantageous in terms of maximizing compliance and vaccine coverage at the same visit. Recently, we sought to generate a vaccine vector by incorporating multiple epitopes into the rotavirus group antigenic protein, VP6. In the present study, a foreign epitope presenting a system using VP6 as a vector was created with six sites on the outer surface of the vector that could be used for insertion of foreign epitopes, and three VP6-based PV1 epitope chimeric proteins were constructed. The chimeric proteins were confirmed by immunoblot, immunofluorescence assay, and injected into guinea pigs to analyze the epitope-specific humoral response. Results showed that these chimeric proteins reacted with anti-VP6F and -PV1 antibodies, and elicited antibodies against both proteins in guinea pigs. Antibodies against the chimeric proteins carrying PV1 epitopes neutralized rotavirus Wa and PV1 infection in vitro. Our study contributes to a better understanding of the use of VP6-based vectors as multiple-epitope delivery vehicles and the epitopes displayed in this form could be considered for development of epitope-based vaccines against rotavirus and poliovirus.

A Simple Proteomics-Based Approach to Identification of Immunodominant Antigens from a Complex Pathogen: Application to the CD4 T Cell Response against Human Herpesvirus 6B.

Most of humanity is chronically infected with human herpesvirus 6 (HHV-6), with viral replication controlled at least in part by a poorly characterized CD4 T cell response. Identification of viral epitopes recognized by CD4 T cells is complicated by the large size of the herpesvirus genome and a low frequency of circulating T cells responding to the virus. Here, we present an alternative to classical epitope mapping approaches used to identify major targets of the T cell response to a complex pathogen like HHV-6B. In the approach presented here, extracellular virus preparations or virus-infected cells are fractionated by SDS-PAGE, and eluted fractions are used as source of antigens to study cytokine responses in direct ex vivo T cell activation studies. Fractions inducing significant cytokine responses are analyzed by mass spectrometry to identify viral proteins, and a subset of peptides from these proteins corresponding to predicted HLA-DR binders is tested for IFN-γ production in seropositive donors with diverse HLA haplotypes. Ten HHV-6B viral proteins were identified as immunodominant antigens. The epitope-specific response to HHV-6B virus was complex and variable between individuals. We identified 107 peptides, each recognized by at least one donor, with each donor having a distinctive footprint. Fourteen peptides showed responses in the majority of donors. Responses to these epitopes were validated using in vitro expanded cells and naturally expressed viral proteins. Predicted peptide binding affinities for the eight HLA-DRB1 alleles investigated here correlated only modestly with the observed CD4 T cell responses. Overall, the response to the virus was dominated by peptides from the major capsid protein U57 and major antigenic protein U11, but responses to other proteins including glycoprotein H (U48) and tegument proteins U54 and U14 also were observed. These results provide a means to follow and potentially modulate the CD4 T-cell immune response to HHV-6B.

Cytomegalovirus-Specific CD4 T Cells Are Cytolytic and Mediate Vaccine Protection.

CD4 T cells provide protection against cytomegalovirus (CMV) and other persistent viruses, and the ability to quantify and characterize epitope-specific responses is essential to gain a more precise understanding of their effector roles in this regard. Here, we report the first two I-A(d)-restricted CD4 T cell responses specific for mouse CMV (MCMV) epitopes and use a major histocompatibility complex class II (MHC-II) tetramer to characterize their phenotypes and functions. We demonstrate that MCMV-specific CD4 T cells can express high levels of granzyme B and kill target cells in an epitope- and organ-specific manner. In addition, CD4 T cell epitope vaccination of immunocompetent mice reduced MCMV replication in the same organs where CD4 cytotoxic T lymphocyte (CTL) activity was observed. Together, our studies show that MCMV epitope-specific CD4 T cells have the potential to mediate antiviral defense by multiple effector mechanisms in vivo. CD4 T cells mediate immune protection by using their T cell receptors to recognize specific portions of viral proteins, called epitopes, that are presented by major histocompatibility complex class II (MHC-II) molecules on the surfaces of professional antigen-presenting cells (APCs). In this study, we discovered the first two epitopes derived from mouse cytomegalovirus (MCMV) that are recognized by CD4 T cells in BALB/c mice, a mouse strain commonly used to study the pathogenesis of this virus infection. Here, we report the sequences of these epitopes, characterize the CD4 T cells that recognize them to fight off MCMV infection, and show that we can use the epitopes to vaccinate mice and protect against MCMV.

A Synthetic Virus-Like Particle Streptococcal Vaccine Candidate Using B-Cell Epitopes from the Proline-Rich Region of Pneumococcal Surface Protein A.

Alternatives to the well-established capsular polysaccharide-based vaccines against Streptococcus pneumoniae that circumvent limitations arising from limited serotype coverage and the emergence of resistance due to capsule switching (serotype replacement) are being widely pursued. Much attention is now focused on the development of recombinant subunit vaccines based on highly conserved pneumococcal surface proteins and virulence factors. A further step might involve focusing the host humoral immune response onto protective protein epitopes using as immunogens structurally optimized epitope mimetics. One approach to deliver such epitope mimetics to the immune system is through the use of synthetic virus-like particles (SVLPs). SVLPs are made from synthetic coiled-coil lipopeptides that are designed to spontaneously self-assemble into 20–30 nm diameter nanoparticles in aqueous buffer. Multivalent display of epitope mimetics on the surface of SVLPs generates highly immunogenic nanoparticles that elicit strong epitope-specific humoral immune responses without the need for external adjuvants. Here, we set out to demonstrate that this approach can yield vaccine candidates able to elicit a protective immune response, using epitopes derived from the proline-rich region of pneumococcal surface protein A (PspA). These streptococcal SVLP-based vaccine candidates are shown to elicit strong humoral immune responses in mice. Following active immunization and challenge with lethal doses of streptococcus, SVLP-based immunogens are able to elicit significant protection in mice. Furthermore, a mimetic-specific monoclonal antibody is shown to mediate partial protection upon passive immunization. The results show that SVLPs combined with synthetic epitope mimetics may have potential for the development of an effective vaccine against Streptococcus pneumoniae.

Presentation of available CTL epitopes that induction of cell-mediated immune response against HIV-1 Koran clade B strain using computational technology.

Theoretical predicting cytotoxic T lymphocyte (CTL) epitopes are an important tool in vaccine design and CTL therapy for enhancing our understanding of the cellular immune system. We would like to identify available CTL epitopes against HIV-1 Korean clade B. CTL activity was assessed in freshly isolated peripheral blood mononuclear cells from Korean HIV patients in order to assess whether these CTL epitopes induce a cell-mediated immune response (CMI). NetCTLpan1.1 software, which is the most popular prediction computer software package, and full atom-based simulation (FABS), which is a 3D modelling system for binding activity between epitopes and human leucocyte antigen (HLA) molecules, were used to predict the peptide-spanning Env region binding to HLA-A*24:02, HLA-A*02:01 and HLA-B*15:01, which are frequently found in the Korean population. Granzyme B and interferon-γ ELISPOT assays were used to determine whether identified CTL epitopes induce CMI. Three HIV-1 Korean clade B-specific Env CTL epitopes were identified: Gp41-RYL and Gp41-RQG are localized within gp41, and Gp120-LLQ within gp120. In in vitro assays using granzyme B ELISPOT, Gp120-LLQ and Gp41-RQG induced epitope-specific CTL responses in HLA-restricted cells. In ex vivo assay using IFN-γ ELISPOT, cell-mediated immune responses to Gp41-RYL were present in 50% of HLA-matched patients, and responses to Gp120-LLQ and Gp41-RQG were found in 33% of HLA-matched patients. In this study, we found that a prediction pipeline for CTL epitopes might be based on the most popular computer prediction software and FABS methods. Our results suggest that these CTL epitopes may provide useful tools and information for the development of a therapeutic vaccine against HIV-1 Korean clade B.

Pep19 drives epitope spreading in periodontitis and periodontitis-associated autoimmune diseases.

Epitope spreading is one of valid mechanisms operating in immunopathological processes of infection-induced autoimmune diseases. We hypothesized that the peptide 19 from Porphyromonas gingivalis heat shock protein (HSP) 60 (Pep19) may be the dominant epitope from which epitope-specific immune response to subdominant epitopes may diversify sequentially into autoimmune responses directed at human neoepitopes in P. gingivalis-induced periodontitis and autoimmune diseases. However, the exact feature and mechanism on how Pep19 may drive epitope spreading into human autoantigens in chronic periodontitis or P. gingivalis-induced experimental periodontitis has not been clarified. The present study was performed with the following specific aims: (i) to delineate retrospectively the features of epitope spreading by human cross-sectional analysis; (ii) to demonstrate prospectively the epitope spreading into new antigenic determinants in an ordered, predictable and sequential manner in experimental periodontitis; and (iii) to clarify the mechanism on how immunization with Pep19 may mobilize helper T cells or elicit B-cell responses to human autoantigens and neoantigen. The study was devised for two independent investigations - a cross-sectional analysis on clinical subjects and a prospective analysis on experimental periodontitis - each being subdivided further into two additional independent observations. Cross-sectional dot immunoblot pattern against a panel of peptides of P.gingivalis HSP60 and human HSP60 was performed among age-dependent healthy subjects and between healthy subjects, patients with chronic periodontitis and patients with autoimmune disease, to identify epitope spreading. A peptide-specific T-cell line was established for phenotype analysis and for proliferation assay to an array of identical peptides. An identical prospective analysis was performed in P.gingivalis-induced experimental periodontitis or in Pep19-immunized mice. Cross-reactivity of anti-Pep19 monoclonal antibody was also investigated. A dominant immune response exclusively to Pep19 prevailed in healthy human subjects (before the age of 40) and mice that persisted in chronic periodontitis and autoimmune diseases without being replaced further by subsequent subdominant epitopes. A sequential epitope spreading provoked by Pep19 to subdominant autoantigen peptide 19 from human HSP60 (Hu19) in most healthy human subjects and mice, and to autoantigen peptide 9 from human HSP60 (Hu9) and neoantigen oxidized low-density lipoprotein (ox-LDL) in P.gingivalis-induced chronic periodontitis and autoimmune diseases could be demonstrated in a reproducible and predictable manner. T-cell proliferative activity to multiple autoantigens Hu19, Hu9 and ox-LDL, and cross-reactivity of anti-Pep19 monoclonal antibody to these epitopes may be proposed as cellular and molecular mechanisms responsible for the phenomenon. Moreover, the predictive value of Pep19 for Hu9 increased remarkably in the disease group when compared with that of the healthy group. Taken together, epitope spreading to Hu19, Hu9 and ox-LDL provoked by Pep19 could be proposed as a solid phenomenon observed in P.gingivalis-induced chronic periodontitis and infection-induced autoimmune diseases in a reproducible and predictable manner. T-cell proliferative activity to these peptides and cross-reactivity of anti-Pep19 antibodies to multiple human autoantigens could be proposed as cellular and molecular mechanisms responsible for this phenomenon.

Vaccination for 2009 pandemic H1N1 influenza A did not induce conserved epitope-specific memory CD8 T cell responses in HIV+ northern Thai children

The influenza virus causes severe illness in susceptible populations, including children and people living with human immunodeficiency virus (HIV). Here, we investigated cell-mediated immune responses (CMI) against influenza CD8 T cell conserved epitopes in HIV-infected (HIV+) northern Thai children following the 2009 pandemic H1N1 influenza A vaccination. Sixty HIV+ children were vaccinated with two doses of the 2009 pandemic influenza vaccine and their CD8T cell responses were assessed. We found no significant differences in the increase of cytokines-producing and CD107a-expressing CD8+ T cells or CD8+ memory T cells in response to pooled conserved epitopes stimulation in vitro between children with different serologic responses to the vaccine at all time points of the study. Our results suggest that the 2009 pandemic H1N1 vaccine did not induce the conserved epitope-specific immune responses in HIV+ children. Vaccine design and vaccination strategy against influenza in these populations warrant further studies.

Different alpha fetoprotein epitope-specific CD8 T cells possess distinct antitumor function (VAC13P.1130)

Abstract Using lentivector (lv) immunization and epitope-optimization, we have recently identified three CD8 epitopes in the hepatocellular carcinoma (HCC) associated antigen of murine alpha fetoprotein (AFP) that could effectively activate CD8 T cells in mice, resulting in potent antitumor effect that can completely prevent mice from tumor challenge and prevent carcinogen-induce autochthonous HCC. In this report, we studied whether the epitope-specific CD8 responses can be further enhanced by peptide boost and investigate if the newly identified epitope specific T cells can kill AFP+ tumor cells and have equal antitumor activity. We found that the lv-primed AFP-specific CD8 responses can be boosted by AFP peptides even at the peak of primary responses. As much as 20 folds increase of AFP-specific CD8 responses can be achieved by peptide boost. However, the kinetics and the antitumor effect of different epitope-specific CD8 responses are drastically different. While the epitope AFP499-specific CD8 T cells possess potent CTL activity against AFP+ tumor cells and generate strong antitumor effect in vivo, the AFP212-specific CD8 T cells have a very limited CTL activity and antitumor effect though they can produce high level of IFNg. The mechanisms underpinning the functional differences will be reported. This finding demonstrates that cancer vaccines must be able to elicit tumor-killing immune effector cells in order to generate antitumor effect.

Loss of Immunization-Induced Epitope-Specific CD4 T-Cell Response following Anaplasma marginale Infection Requires Presence of the T-Cell Epitope on the Pathogen and Is Not Associated with an Increase in Lymphocytes Expressing Known Regulatory Cell Phenotypes.

We have shown that in cattle previously immunized with outer membrane proteins, infection with Anaplasma marginale induces a functionally exhausted CD4 T-cell response to the A. marginale immunogen. Furthermore, T-cell responses following infection in nonimmunized cattle had a delayed onset and were sporadic and transient during persistent infection. The induction of an exhausted T-cell response following infection presumably facilitates pathogen persistence. In the current study, we hypothesized that the loss of epitope-specific T-cell responses requires the presence of the immunizing epitope on the pathogen, and T-cell dysfunction correlates with the appearance of regulatory T cells. In limited studies in cattle, regulatory T cells have been shown to belong to γδ T-cell subsets rather than be CD4 T cells expressing forkhead box protein P3 (FoxP3). Cattle expressing the DRB3*1101 haplotype were immunized with a truncated A. marginale major surface protein (MSP) 1a that contains a DRB3*1101-restricted CD4 T-cell epitope, F2-5B. Cattle either remained unchallenged or were challenged with A. marginale bacteria that express the epitope or with A. marginale subsp. centrale that do not. Peripheral blood and spleen mononuclear cells were monitored for MSP1a epitope F2-5B-specfic T-cell proliferative responses and were stained for γδ T-cell subsets or CD4(+) CD25(+) FoxP3(+) T cells before and during infection. As hypothesized, the induction of T-cell exhaustion occurred only following infection with A. marginale, which did not correlate with an increase in either CD4(+) CD25(+) FoxP3(+) T cells or any γδ T-cell subset examined.

Frequencies of Gag-restricted T-cell escape “footprints” differ across HIV-1 clades A1 and D chronically infected Ugandans irrespective of host HLA B alleles

Objective(s)We evaluated relationships between critical Gag T-cell escape mutations and concomitant T-cell responses to determine whether HLA-restricted Gag mutations that confer protection, occur at similar rates in a population infected with mixed HIV-1 clades A1 and D viruses. MethodsAssessment of Gag selective pressure, and adaptive T-cell functions to KAFSPEVIPMF (KF11), ISPRTLNAW (ISW9) and TSTLQEQIGW (TW10) Gag epitopes were combined with host HLA to assess correlations with rates of critical epitope escape mutations in clades A1- (n=23) and D- (n=21) infected, untreated subjects. Infecting clades and selection pressure were determined from the gag sequences. ResultsOverall, Gag escape mutations A163X in KF11 were detected in 61% (14/23) A1- infected compared to 5% (1/21) in D-infected subjects (p=0.00015). Gag mutations I147X in the ISW9 epitope were seen in 43%: (10/23) clade A compared to 5%: (1/21) clade D infected subjects, p=0.007, Fisher's Exact test. Both mutations were more frequent in clade A1 infection. Frequencies of the measured epitope-specific T-cell responses were comparable across clades. Peptide binding affinities for the restricting HLA alleles did not differ across clades. Overall, selection pressure on the Gag protein was significantly greater in clade A than in clade D sequences. ConclusionsThese findings imply that HIV-1 vaccine strategies designed to target structurally constrained T-cell epitopes may be further challenged by clade-driven outcomes in specific HLA-restricted Gag epitopes. Equally, the data are line with slower HIV-1 disease progression in clade A infection; and raise hope that increased selective pressure on Gag may be protective irrespective of host HLA alleles.

CD4 T cells specific for a latency-associated γ-herpesvirus epitope are polyfunctional and cytotoxic.

The oncogenic γ-herpesviruses EBV and Kaposi sarcoma-associated herpesvirus are ubiquitous human pathogens that establish lifelong latent infections maintained by intermittent viral reactivation and reinfection. Effector CD4 T cells are critical for control of viral latency and in immune therapies for virus-associated tumors. In this study, we exploited γHV68 infection of mice to enhance our understanding of the CD4 T cell response during γ-herpesvirus infection. Using a consensus prediction approach, we identified 16 new CD4 epitope-specific responses that arise during lytic infection. An additional epitope encoded by the M2 protein induced uniquely latency-associated CD4 T cells, which were not detected at the peak of lytic infection but only during latency and were not induced postinfection with a latency-deficient virus. M2-specific CD4 T cells were selectively cytotoxic, produced multiple antiviral cytokines, and sustained IL-2 production. Identification of latency-associated cytolytic CD4 T cells will aid in dissecting mechanisms of CD4 immune control of γ-herpesvirus latency and the development of therapeutic approaches to control viral reactivation and pathology.

Group A Streptococcal vaccine candidate: contribution of epitope to size, antigen presenting cell interaction and immunogenicity.

Utilize lipopeptide vaccine delivery system to develop a vaccine candidate against Group A Streptococcus. Lipopeptides synthesized by solid-phase peptide synthesis-bearing carboxyl (C)-terminal and amino (N)-terminal Group A Streptococcus peptide epitopes. Nanoparticles formed were evaluated in vivo. Immune responses were induced in mice without additional adjuvant. We demonstrated for the first time that incorporation of the C-terminal epitope significantly enhanced the N-terminal epitope-specific antibody response and correlated with forming smaller nanoparticles. Antigen-presenting cells had increased uptake and maturation by smaller, more immunogenic nanoparticles. Antibodies raised by vaccination recognized isolates. Demonstrated the lipopeptidic nanoparticles to induce an immune response which can be influenced by the combined effect of epitope choice and size.

Emergence of broadly neutralizing antibodies and viral coevolution in two subjects during the early stages of infection with human immunodeficiency virus type 1.

Delineating the key early events that lead to the development of broadly neutralizing anti-HIV-1 antibodies during natural infection may help guide the development of immunogens and vaccine regimens to prevent HIV-1 infection. In this study, we monitored two HIV-1-positive subjects, VC20013 and VC10014, over the course of infection from before they developed broadly neutralizing antibody (bNAb) activity until several years after neutralizing breadth was detected in plasma. Both subjects developed bNAb activity after approximately 1 year postinfection, which ultimately mapped to the membrane-proximal external region (MPER) in VC20013 and an epitope that overlaps the CD4 receptor binding site in VC10014. In subject VC20013, we were able to identify anti-MPER activity in the earliest plasma sample that exhibited no bNAb activity, indicating that this epitope specificity was acquired very early on, but that it was initially not able to mediate neutralization. Escape mutations within the bNAb epitopes did not arise in the circulating envelopes until bNAb activity was detectable in plasma, indicating that this early response was not sufficient to drive viral escape. As bNAb activity began to emerge in both subjects, we observed a simultaneous increase in autologous antienvelope antibody binding affinity, indicating that antibody maturation was occurring as breadth was developing. Our findings illustrate one potential mechanism by which bNAbs develop during natural infection in which an epitope target is acquired very early on during the course of infection but require time and maturation to develop into broadly neutralizing activity. One major goal of HIV-1 vaccine research is the development of a vaccine that can elicit broadly neutralizing antibodies (bNAbs). Although no such vaccine exists, bNAbs develop in approximately 20% of HIV-1-infected subjects, providing a prototype of the bNAbs that must be reelicited by vaccine. Thus, there is significant interest in understanding the mechanisms by which bNAbs develop during the course of infection. We studied the timing, epitope specificity, and evolution of the bNAb responses in two HIV-1-positive patients who developed bNAb activity within the first several years after infection. In one subject, antibodies to a broadly neutralizing epitope developed very early but were nonneutralizing. After several months, neutralizing activity developed, and the virus mutated to escape their activity. Our study highlights one mechanism for the development of bNAbs where early epitope acquisition followed by sufficient time for antibody maturation drives the epitope-specific antibody response toward broadly neutralizing activity.