Molecular characterization of HIV-1 CRF01_AE in Mekong Delta, Vietnam, and impact of T-cell epitope mutations on HLA recognition (ANRS 12159)

Background

To date, 11 HIV-1 subtypes and 48 circulating recombinant forms have been described worldwide. The underlying reason why their distribution is so heterogeneous is not clear. Host genetic factors could partly explain this distribution. The aim of this study was to describe HIV-1 strains circulating in an unexplored area of Mekong Delta, Vietnam, and to assess the impact of optimal epitope mutations on HLA binding.

Methods

We recruited 125 chronically antiretroviral-naive HIV-1-infected subjects from five cities in the Mekong Delta. We performed high-resolution DNA typing of HLA class I alleles, sequencing of Gag and RT-Prot genes and phylogenetic analysis of the strains. Epitope mutations were analyzed in patients bearing the HLA allele restricting the studied epitope. Optimal wild-type epitopes from the Los Alamos database were used as reference. T-cell epitope recognition was predicted using the immune epitope database tool according to three different scores involved in antigen processing (TAP and proteasome scores) and HLA binding (MHC score).

Results

All sequences clustered with CRF01_AE. HLA class I genotyping showed the predominance of Asian alleles as A*11:01 and B*46:01 with a Vietnamese specificity held by two different haplotypes. The percentage of homology between Mekong and B consensus HIV-1 sequences was above 85%. Divergent epitopes had TAP and proteasome scores comparable with wild-type epitopes. MHC scores were significantly lower in divergent epitopes with a mean of 2.4 (±0.9) versus 2 (±0.7) in non-divergent ones (p<0.0001).

Conclusions

Our study confirms the wide predominance of CRF01_AE in the Mekong Delta where patients harbor a specific HLA pattern. Moreover, it demonstrates the lower MHC binding affinity among divergent epitopes. This weak immune pressure combined with a narrow genetic diversity favors immune escape and could explain why CRF01_AE is still predominant in Vietnam, particularly in the Mekong area.     

The Effect of HLA Polymorphisms on the Recognition of Gag Epitopes in HIV-1 CRF01_AE Infection

Introduction

The design of a globally effective vaccine rests on the identification of epitopes capable of eliciting effective cytotoxic T lymphocyte (CTL) responses across multiple HIV clades in different populations. This study aims to discern the effect of HLA polymorphisms and the cross-clade reactivity or clade-specificity of epitopes in Thailand where HIV-1 CRF01_AE is circulating.

Materials and Methods

14 peptides based on consensus HIV-1 CRF01_AE amino acid sequences were designed for use in IFN-γ ELISpot assays and ⁵¹Cr release assays among 66 HIV-1 CRF01_AE-infected Thai patients. For ELISpot responders carrying HLA alleles currently unknown to restrict CRF01_AE epitopes, in silico epitope-HLA prediction was performed.

Results

29/66 (43.9%) patients recognized at least one peptide. In total 79 responses were seen against all 14 peptides. 28/79 (35.4%) of the responses were in patients with HLA alleles previously reported to restrict CRF01_AE epitopes, 24/79 (30.4%) responses were in individuals with HLA alleles previously reported to restrict epitopes of HIV clades other than CRF01_AE, and the remaining 27/79 (34.2%) responses were not associated with HLA alleles previously known to restrict HIV epitopes. In silico epitope prediction detected 19 novel, epitope-HLA combinations, and 11/19 (57.9%) were associated with HLA-C alleles. We further confirmed a novel HLA restriction of a previously identified HIV-1 Gag epitope [p24_122–130: PPIPVGDIY (PY9)] by HLA-B*40:01 with a standard ⁵¹Cr release assay.

Discussion

CTL recognition sites in HIV-1 Gag were similar among different clades but the HLA restriction differed in Thai patients. This disparity in HLA restriction along different populations illustrated the importance of clade- and population-specific HLA analysis prior to CTL vaccine design.     

A double-blind randomized phase I clinical trial targeting ALVAC-HIV vaccine to human dendritic cells

Background

We conducted a novel pilot study comparing different delivery routes of ALVAC-HIV (vCP205), a canarypox vaccine containing HIV gene inserts: env, gag and pol. We explored the concept that direct ex vivo targeting of human dendritic cells (DC) would enhance the immune response compared to either conventional intramuscular or intradermal injections of the vaccine alone.

Methodology/Principal Findings

Healthy HIV-1 uninfected volunteers were administered ALVAC-HIV or placebo by intramuscular injection (IM), intradermal injection (ID) or subcutaneous injection (SQ) of autologous ex vivo transfected DC at months 0, 1, 3 and 6. All vaccine delivery routes were well tolerated. Binding antibodies were observed to both the ALVAC vector and HIV-1 gp160 proteins. Modest cellular responses were observed in 2/7 individuals in the DC arm and 1/8 in the IM arm as determined by IFN-γ ELISPOT. Proliferative responses were most frequent in the DC arm where 4/7 individuals had measurable responses to multiple HIV-1 antigens. Loading DC after maturation resulted in lower gene expression, but overall better responses to both HIV-1 and control antigens, and were associated with better IL-2, TNF-α and IFN-γ production.

Conclusions/Significance

ALVAC-HIV delivered IM, ID or SQ with autologous ex vivo transfected DC proved to be safe. The DC arm was most immunogenic. Proliferative immune responses were readily detected with only modest cytotoxic CD8 T cell responses. Loading mature DC with the live viral vaccine induced stronger immune responses than loading immature DC, despite increased transgene expression with the latter approach. Volunteers who received the autologous vaccine loaded mature DC developed a broader and durable immune response compared to those vaccinated by conventional routes.

Trial Registration

ClinicalTrials.gov {"type":"clinical-trial","attrs":{"text":"NCT00013572","term_id":"NCT00013572"}}NCT00013572     

Autologous HIV-1 Clade-B Nef Peptides Elicit Increased Frequency,Breadth and Function of CD8+ T-Cells Compared to Consensus Peptides

Objective

To determine the function and phenotype of CD8⁺ T-cells targeting consensus and autologous sequences of entire HIV-1 Nef protein.

Methods

Multiparameter flow cytometry-based analysis was used to evaluate the responses of two treatment naïve HIV-infected individuals, during primary and the chronic phases of infection.

Results

A greater breadth and magnitude of CD8 IFN-γ responses to autologous compared to clade-B consensus peptides was observed in both subjects. Cross recognition between autologous and consensus peptides decreased in both subjects during progression from primary to chronic infection. The frequencies of TEMRA and TEM CD8⁺ T-cells targeting autologous peptides were higher than those targeting consensus peptides and were more polyfunctional (IFN-γ⁺ Gr-B⁺ CD107a⁺).

Conclusions

Our data indicate superior sensitivity and specificity of autologous peptides. The functional and maturational aspects of “real” versus “cross-recognized” responses were also found to differ, highlighting the importance of a sequence-specific approach towards understanding HIV immune response.     

Broad and Gag-biased HIV-1 epitope repertoires are associated with lower viral loads

Background

HLA class-I alleles differ in their ability to control HIV replication through cell-mediated immune responses. No consistent associations have been found between the breadth of Cytotoxic T Lymphocytes (CTL) responses and the control of HIV-1, and it is unknown whether the size or distribution of the viral proteome-wide epitope repertoire, i.e., the intrinsic ability to present fewer, more or specific viral epitopes, could affect clinical markers of disease progression.

Methodology/Principal Findings

We used an epitope prediction model to identify all epitope motifs in a set of 302 HIV-1 full-length proteomes according to each individual''s HLA (Human Leukocyte Antigen) genotype. The epitope repertoire, i.e., the number of predicted epitopes per HIV-1 proteome, varied considerably between HLA alleles and thus among individual proteomes. In a subgroup of 270 chronically infected individuals, we found that lower viral loads and higher CD4 counts were associated with a larger predicted epitope repertoire. Additionally, in Gag and Rev only, more epitopes were restricted by alleles associated with low viral loads than by alleles associated with higher viral loads.

Conclusions/Significance

This comprehensive analysis puts forth the epitope repertoire as a mechanistic component of the multi-faceted HIV-specific CTL response. The favorable impact on markers of disease status of the propensity to present more HLA binding peptides and specific proteins gives impetus to vaccine design strategies that seek to elicit responses to a broad array of HIV-1 epitopes, and suggest a particular focus on Gag.     

Development of a Novel In Silico Docking Simulation Model for the Fine HIV-1 Cytotoxic T Lymphocyte Epitope Mapping

Introduction

Class I HLA''s polymorphism has hampered CTL epitope mapping with laborious experiments. Objectives are 1) to evaluate the novel in silico model in predicting previously reported epitopes in comparison with existing program, and 2) to apply the model to predict optimal epitopes with HLA using experimental results.

Materials and Methods

We have developed a novel in silico epitope prediction method, based on HLA crystal structure and a peptide docking simulation model, calculating the peptide-HLA binding affinity at four amino acid residues in each terminal. It was applied to predict 52 HIV best–defined CTL epitopes from 15-mer overlapping peptides, and its predictive ability was compared with the HLA binding motif-based program of HLArestrictor. It was then used to predict HIV-1 Gag optimal epitopes from previous ELISpot results.

Results

43/52 (82.7%) epitopes were detected by the novel model, whereas 37 (71.2%) by HLArestrictor. We also found a significant reduction in epitope detection rates for longer epitopes in HLArestrictor (p = 0.027), but not in the novel model. Improved epitope prediction was also found by introducing both models, especially in specificity (p<0.001). Eight peptides were predicted as novel, immunodominant epitopes in both models.

Discussion

This novel model can predict optimal CTL epitopes, which were not detected by an existing program. This model is potentially useful not only for narrowing down optimal epitopes, but predicting rare HLA alleles with less information. By introducing different principal models, epitope prediction will be more precise.     

A Novel Liposome-Based Adjuvant CAF01 for Induction of CD8+ Cytotoxic T-Lymphocytes (CTL) to HIV-1 Minimal CTL Peptides in HLA-A*0201 Transgenic Mice

Background

Specific cellular cytotoxic immune responses (CTL) are important in combating viral diseases and a highly desirable feature in the development of targeted HIV vaccines. Adjuvants are key components in vaccines and may assist the HIV immunogens in inducing the desired CTL responses. In search for appropriate adjuvants for CD8⁺ T cells it is important to measure the necessary immunological features e.g. functional cell killing/lysis in addition to immunological markers that can be monitored by simple immunological laboratory methods.

Methodology/Principal Findings

We tested the ability of a novel two component adjuvant, CAF01, consisting of the immune stimulating synthetic glycolipid TDB (Trehalose-Dibehenate) incorporated into cationic DDA (Dimethyldioctadecylammonium bromide) liposomes to induce CD8⁺ T-cell restricted cellular immune responses towards subdominant minimal HLA-A0201-restricted CTL epitopes from HIV-1 proteins in HLA-A*0201 transgenic HHD mice. CAF01 has an acceptable safety profile and is used in preclinical development of vaccines against HIV-1, malaria and tuberculosis.

Conclusions/Significance

We found that CAF01 induced cellular immune responses against HIV-1 minimal CTL epitopes in HLA-A*0201 transgenic mice to levels comparable with that of incomplete Freund''s adjuvant.     

Epitope Mapping of HIV-Specific CD8+ T cells in a Cohort Dominated by Clade A1 Infection

Background

CD8+ T cell responses are often detected at large magnitudes in HIV-infected subjects, and eliciting these responses is the central aim of many HIV-1 vaccine strategies. Population differences in CD8+ T cell epitope specificity will need to be understood if vaccines are to be effective in multiple geographic regions.

Methodology/Principal Findings

In a large Kenyan cohort, we compared responsive CD8+ T cell HIV-1 Env overlapping peptides (OLPs) to Best Defined Epitopes (BDEs), many of which have been defined in clade B infection. While the majority of BDEs (69%) were recognized in this population, nearly half of responsive OLPs (47%) did not contain described epitopes. Recognition frequencies of BDEs were inversely correlated to epitopic sequence differences between clade A1 and BDE (P = 0.019), and positively selected residues were more frequent in “new” OLPs (without BDEs). We assessed the impact of HLA and TAP binding on epitope recognition frequencies, focusing on predicted and actual epitopes in the HLA B7 supertype.

Conclusions/Significance

Although many previously described CD8 epitopes were recognized, several novel CD8 epitopes were defined in this population, implying that epitope mapping efforts have not been completely exhausted. Expansion of these studies will be critical to understand population differences in CD8 epitope recognition.     

Direct phenotypical and functional dysregulation of primary human B cells by human immunodeficiency virus (HIV) type 1 in vitro

Background

Human immunodeficiency virus type 1 (HIV-1) induces a general dysregulation of immune system. Dysregulation of B cell compartment is generally thought to be induced by HIV-related immune activation and lymphopenia. However, a direct influence of HIV-1 particles on B cells was recently proposed as the third pathway of B cells dysregulation.

Methods/Principal Findings

We evaluated the direct and specific consequences of HIV-1 contact on activation, survival, proliferation and phenotype of primary B cells in vitro. Moreover, we examined expression of activation-induced cytidine deaminase (AID) mRNA that is responsible for class switch recombination (CSR) and somatic hypermutation (SHM). Here, we report that changes observed in cellular proliferation, phenotypes and activation of B cells could be caused by direct contact between HIV-1 particles and primary B cells in vitro. Finally, direct HIV-1-derived B cells activation led to the increase of AID mRNA expression and its subsequent CSR function was detected in vitro.

Conclusion/Significance

We showed that HIV-1 could directly induce primary B cells dysregulation triggering phenotypical and functional abilities of B cells in vitro that could explain in some extent early B-cell abnormalities in HIV disease.     

Comparison of HIV-1 nef and gag Variations and Host HLA Characteristics as Determinants of Disease Progression among HIV-1 Vertically Infected Kenyan Children

Objectives

Disease progression varies among HIV-1-infected individuals. The present study aimed to explore possible viral and host factors affecting disease progression in HIV-1-infected children.

Methods

Since 2000, 102 HIV-1 vertically-infected children have been followed-up in Kenya. Here we studied 29 children (15 male/14 female) who started antiretroviral treatment at <5 years of age (rapid progressors; RP), and 32 (17 male/15 female) who started at >10 years of age (slow progressors; SP). Sequence variations in the HIV-1 gag and nef genes and the HLA class I-related epitopes were compared between the two groups.

Results

Based on nef sequences, HIV-1 subtypes A1/D were detected in 62.5%/12.5% of RP and 66.7%/20% of SP, with no significant difference in subtype distribution between groups (p = 0.8). In the ten Nef functional domains, only the PxxP₃ region showed significantly greater variation in RP (33.3%) than SP (7.7%, p = 0.048). Gag sequences did not significantly differ between groups. The reportedly protective HLA-A alleles, A*74:01, A*32:01 and A*26, were more commonly observed in SP (50.0%) than RP (11.1%, p = 0.010), whereas the reportedly disease-susceptible HLA-B*45:01 was more common in RP (33.3%) than SP (7.4%, p = 0.045). Compared to RP, SP showed a significantly higher median number of predicted HLA-B-related 12-mer epitopes in Nef (3 vs. 2, p = 0.037), HLA-B-related 11-mer epitopes in Gag (2 vs. 1, p = 0.029), and HLA-A-related 9-mer epitopes in Gag (4 vs. 1, p = 0.051). SP also had fewer HLA-C-related epitopes in Nef (median 4 vs. 5, p = 0.046) and HLA-C-related 11-mer epitopes in Gag (median 1 vs. 1.5, p = 0.044) than RP.

Conclusions

Compared to rapid progressors, slow progressors had more protective HLA-A alleles and more HLA-B-related epitopes in both the Nef and Gag proteins. These results suggest that the host factor HLA plays a stronger role in disease progression than the Nef and Gag sequence variations in HIV-1-infected Kenyan children.     

T-cell immune responses against Env from CRF12_BF and subtype B HIV-1 show high clade-specificity that can be overridden by multiclade immunizations

Background

The extreme genetic diversity of the human immunodeficiency virus type 1 (HIV-1) poses a daunting challenge to the generation of an effective AIDS vaccine. In Argentina, the epidemic is characterized by the high prevalence of infections caused by subtype B and BF variants. The aim of this study was to characterize in mice the immunogenic and antigenic properties of the Env protein from CRF12_BF in comparison with clade B, employing prime-boost schemes with the combination of recombinant DNA and vaccinia virus (VV) vectors.

Methodology/Principal Findings

As determined by ELISPOT from splenocytes of animals immunized with either EnvBF or EnvB antigens, the majority of the cellular responses to Env were found to be clade-specific. A detailed peptide mapping of the responses reveal that when there is cross-reactivity, there are no amino acid changes in the peptide sequence or were minimal and located at the peptide ends. In those cases, analysis of T cell polifunctionality and affinity indicated no differences with respect to the cellular responses found against the original homologous sequence.Significantly, application of a mixed immunization combining both clades (B and BF) induced a broader cellular response, in which the majority of the peptides targeted after the single clade vaccinations generated a positive response. In this group we could also find significant cellular and humoral responses against the whole gp120 protein from subtype B.

Conclusions/Significance

This work has characterized for the first time the immunogenic peptides of certain EnvBF regions, involved in T cell responses. It provides evidence that to improve immune responses to HIV there is a need to combine Env antigens from different clades, highlighting the convenience of the inclusion of BF antigens in future vaccines for geographic regions where these HIV variants circulate.     

Immunogenic Profiling in Mice of a HIV/AIDS Vaccine Candidate (MVA-B) Expressing Four HIV-1 Antigens and Potentiation by Specific Gene Deletions

Background

The immune parameters of HIV/AIDS vaccine candidates that might be relevant in protection against HIV-1 infection are still undefined. The highly attenuated poxvirus strain MVA is one of the most promising vectors to be use as HIV-1 vaccine. We have previously described a recombinant MVA expressing HIV-1 Env, Gag, Pol and Nef antigens from clade B (referred as MVA-B), that induced HIV-1-specific immune responses in different animal models and gene signatures in human dendritic cells (DCs) with immunoregulatory function.

Methodology/Principal Findings

In an effort to characterize in more detail the immunogenic profile of MVA-B and to improve its immunogenicity we have generated a new vector lacking two genes (A41L and B16R), known to counteract host immune responses by blocking the action of CC-chemokines and of interleukin 1β, respectively (referred as MVA-B ΔA41L/ΔB16R). A DNA prime/MVA boost immunization protocol was used to compare the adaptive and memory HIV-1 specific immune responses induced in mice by the parental MVA-B and by the double deletion mutant MVA-B ΔA41L/ΔB16R. Flow cytometry analysis revealed that both vectors triggered HIV-1-specific CD4⁺ and CD8⁺ T cells, with the CD8⁺ T-cell compartment responsible for >91.9% of the total HIV-1 responses in both immunization groups. However, MVA-B ΔA41L/ΔB16R enhanced the magnitude and polyfunctionality of the HIV-1-specific CD4⁺ and CD8⁺ T-cell immune responses. HIV-1-specific CD4⁺ T-cell responses were polyfunctional and preferentially Env-specific in both immunization groups. Significantly, while MVA-B induced preferentially Env-specific CD8⁺ T-cell responses, MVA-B ΔA41L/ΔB16R induced more GPN-specific CD8⁺ T-cell responses, with an enhanced polyfunctional pattern. Both vectors were capable of producing similar levels of antibodies against Env.

Conclusions/Significance

These findings revealed that MVA-B and MVA-B ΔA41L/ΔB16R induced in mice robust, polyfunctional and durable T-cell responses to HIV-1 antigens, but the double deletion mutant showed enhanced magnitude and quality of HIV-1 adaptive and memory responses. Our observations are relevant in the immune evaluation of MVA-B and on improvements of MVA vectors as HIV-1 vaccines.     

Equivalent T Cell Epitope Promiscuity in Ecologically Diverse Human Pathogens

Background

The HLA (human leukocyte antigen) molecules that present pathogen-derived epitopes to T cells are highly diverse. Correspondingly, many pathogens such as HIV evolve epitope variants in order to evade immune recognition. In contrast, another persistent human pathogen, Mycobacterium tuberculosis, has highly conserved epitope sequences. This raises the question whether there is also a difference in the ability of these pathogens’ epitopes to bind diverse HLA alleles, referred to as an epitope’s binding promiscuity. To address this question, we compared the in silico HLA binding promiscuity of T cell epitopes from pathogens with distinct infection strategies and outcomes of human exposure.

Methods

We used computer algorithms to predict the binding affinity of experimentally-verified microbial epitope peptides to diverse HLA-DR, HLA-A and HLA-B alleles. We then analyzed binding promiscuity of epitopes derived from HIV and M. tuberculosis. We also analyzed promiscuity of epitopes from Streptococcus pyogenes, which is known to exhibit epitope diversity, and epitopes of Bacillus anthracis and Clostridium tetani toxins, as these bacteria do not depend on human hosts for their survival or replication, and their toxin antigens are highly immunogenic human vaccines.

Results

We found that B. anthracis and C. tetani epitopes were the most promiscuous of the group that we analyzed. However, there was no consistent difference or trend in promiscuity in epitopes contained in HIV, M. tuberculosis, and S. pyogenes.

Conclusions

Our results show that human pathogens with distinct immune evasion strategies and epitope diversities exhibit equivalent levels of T cell epitope promiscuity. These results indicate that differences in epitope promiscuity do not account for the observed differences in epitope variation and conservation.     

HLA Class I-T cell epitopes from trans-sialidase proteins reveal functionally distinct subsets of CD8+ T cells in chronic Chagas disease

Background

Previously, we identified a set of HLA-A020.1-restricted trans-sialidase peptides as targets of CD8⁺ T cell responses in HLA-A0201⁺ individuals chronically infected by T. cruzi.

Methods and Findings

Herein, we report the identification of peptides encoded by the same trans-sialidase gene family that bind alleles representative of the 6 most common class I HLA-supertypes. Based on a combination of bioinformatic predictions and HLA-supertype considerations, a total of 1001 epitopes predicted to bind to HLA A01, A02, A03, A24, B7 and B44 supertypes was selected. Ninety-six supertype-binder epitopes encoded by multiple trans-sialidase genes were tested for the ability to stimulate a recall CD8⁺ T cell response in the peripheral blood from subjects with chronic T. cruzi infection regardless the HLA haplotype. An overall hierarchy of antigenicity was apparent, with the A02 supertype peptides being the most frequently recognized in the Chagas disease population followed by the A03 and the A24 supertype epitopes. CD8⁺ T cell responses to promiscuous epitopes revealed that the CD8⁺ T cell compartment specific for T. cruzi displays a functional profile with T cells secreting interferon-γ alone as the predominant pattern and very low prevalence of single IL-2-secreting or dual IFN-γ/IL-2 secreting T cells denoting a lack of polyfunctional cytokine responses in chronic T. cruzi infection.

Conclusions

This study identifies a set of T. cruzi peptides that should prove useful for monitoring immune competence and changes in infection and disease status in individuals with chronic Chagas disease.     

Identification of CD8+ T Cell Epitopes in the West Nile Virus Polyprotein by Reverse-Immunology Using NetCTL

Background

West Nile virus (WNV) is a growing threat to public health and a greater understanding of the immune response raised against WNV is important for the development of prophylactic and therapeutic strategies.

Methodology/Principal Findings

In a reverse-immunology approach, we used bioinformatics methods to predict WNV-specific CD8⁺ T cell epitopes and selected a set of peptides that constitutes maximum coverage of 20 fully-sequenced WNV strains. We then tested these putative epitopes for cellular reactivity in a cohort of WNV-infected patients. We identified 26 new CD8⁺ T cell epitopes, which we propose are restricted by 11 different HLA class I alleles. Aiming for optimal coverage of human populations, we suggest that 11 of these new WNV epitopes would be sufficient to cover from 48% to 93% of ethnic populations in various areas of the World.

Conclusions/Significance

The 26 identified CD8⁺ T cell epitopes contribute to our knowledge of the immune response against WNV infection and greatly extend the list of known WNV CD8⁺ T cell epitopes. A polytope incorporating these and other epitopes could possibly serve as the basis for a WNV vaccine.     

Insertion of Vaccinia Virus C7L Host Range Gene into NYVAC-B Genome Potentiates Immune Responses against HIV-1 Antigens

Background

The highly attenuated vaccinia virus strain NYVAC expressing HIV-1 components has been evaluated as a vaccine candidate in preclinical and clinical trials with encouraging results. We have previously described that the presence of C7L in the NYVAC genome prevents the induction of apoptosis and renders the vector capable of replication in human and murine cell lines while maintaining an attenuated phenotype in mice.

Methodology/Principal Findings

In an effort to improve the immunogenicity of NYVAC, we have developed a novel poxvirus vector by inserting the VACV host-range C7L gene into the genome of NYVAC-B, a recombinant virus that expresses four HIV-1 antigens from clade B (Env, Gag, Pol and Nef) (referred as NYVAC-B-C7L). In the present study, we have compared the in vitro and in vivo behavior of NYVAC-B and NYVAC-B-C7L. In cultured cells, NYVAC-B-C7L expresses higher levels of heterologous antigen than NYVAC-B as determined by Western blot and fluorescent-activated cell sorting to score Gag expressing cells. In a DNA prime/poxvirus boost approach with BALB/c mice, both recombinants elicited robust, broad and multifunctional antigen-specific T-cell responses to the HIV-1 immunogens expressed from the vectors. However, the use of NYVAC-B-C7L as booster significantly enhanced the magnitude of the T cell responses, and induced a more balanced cellular immune response to the HIV-1 antigens in comparison to that elicited in animals boosted with NYVAC-B.

Conclusions/Significance

These findings demonstrate the possibility to enhance the immunogenicity of the highly attenuated NYVAC vector by the insertion of the host-range gene C7L and suggest the use of this modified vector as an improved vaccine candidate against HIV/AIDS.     

A New Adenovirus Based Vaccine Vector Expressing an Eimeria tenella Derived TLR Agonist Improves Cellular Immune Responses to an Antigenic Target

Background

Adenoviral based vectors remain promising vaccine platforms for use against numerous pathogens, including HIV. Recent vaccine trials utilizing Adenovirus based vaccines expressing HIV antigens confirmed induction of cellular immune responses, but these responses failed to prevent HIV infections in vaccinees. This illustrates the need to develop vaccine formulations capable of generating more potent T-cell responses to HIV antigens, such as HIV-Gag, since robust immune responses to this antigen correlate with improved outcomes in long-term non-progressor HIV infected individuals.

Methodology/Principal Findings

In this study we designed a novel vaccine strategy utilizing an Ad-based vector expressing a potent TLR agonist derived from Eimeria tenella as an adjuvant to improve immune responses from a [E1-]Ad-based HIV-Gag vaccine. Our results confirm that expression of rEA elicits significantly increased TLR mediated innate immune responses as measured by the influx of plasma cytokines and chemokines, and activation of innate immune responding cells. Furthermore, our data show that the quantity and quality of HIV-Gag specific CD8⁺ and CD8⁻ T-cell responses were significantly improved when coupled with rEA expression. These responses also correlated with a significantly increased number of HIV-Gag derived epitopes being recognized by host T cells. Finally, functional assays confirmed that rEA expression significantly improved antigen specific CTL responses, in vivo. Moreover, we show that these improved responses were dependent upon improved TLR pathway interactions.

Conclusion/Significance

The data presented in this study illustrate the potential utility of Ad-based vectors expressing TLR agonists to improve clinical outcomes dependent upon induction of robust, antigen specific immune responses.     

HLA-Associated Immune Pressure on Gag Protein in CRF01_AE-Infected Individuals and Its Association with Plasma Viral Load

Background

The human leukocyte antigen (HLA)-restricted cytotoxic T-lymphocyte (CTL) immune response is one of the major factors determining the genetic diversity of human immunodeficiency virus (HIV). There are few population-based analyses of the amino acid variations associated with the host HLA type and their clinical relevance for the Asian population. Here, we identified HLA-associated polymorphisms in the HIV-1 CRF01_AE Gag protein in infected married couples, and examined the consequences of these HLA-selected mutations after transmission to HLA-unmatched recipients.

Methodology/Principal Findings

One hundred sixteen HIV-1-infected couples were recruited at a government hospital in northern Thailand. The 1.7-kb gag gene was amplified and directly sequenced. We identified 56 associations between amino acid variations in Gag and HLA alleles. Of those amino acid variations, 35 (62.5%) were located within or adjacent to regions reported to be HIV-specific CTL epitopes restricted by the relevant HLA. Interestingly, a significant number of HLA-associated amino acid variations appear to be unique to the CRF01_AE-infected Thai population. Variations in the capsid protein (p24) had the strongest associations with the viral load and CD4 cell count. The mutation and reversion rates after transmission to a host with a different HLA environment varied considerably. The p24 T242N variant escape from B57/58 CTL had a significant impact on the HIV-1 viral load of CRF01_AE-infected patients.

Conclusions/Significance

HLA-associated amino acid mutations and the CTL selection pressures on the p24 antigen appear to have the most significant impact on HIV replication in a CRF01_AE-infected Asian population. HLA-associated mutations with a low reversion rate accumulated as a footprint in this Thai population. The novel HLA-associated mutations identified in this study encourage us to acquire more extensive information about the viral dynamics of HLA-associated amino acid polymorphisms in a given population as effective CTL vaccine targets.     

Evaluation of OspA Vaccination-Induced Serological Correlates of Protection against Lyme Borreliosis in a Mouse Model

Background

For clinical development of a novel multivalent OspA vaccine against Lyme borreliosis, serological assays are required which can be used to establish immune correlates of protection against infection with Borrelia.

Methods

Four assays (an OspA IgG ELISA, a competitive inhibition (CI) ELISA, a Borrelia surface-binding (SB) assay and a Borrelia killing assay) were used to evaluate the correlation between immune responses induced by rOspA 1/2 (a chimeric immunogen containing protective epitopes from OspA serotypes 1 and 2), and protective immunity against infection by B. burgdorferi s.s. (OspA-1) and B. afzelii (OspA-2). Mice were immunized with OspA 1/2 doses ranging from 0.3 ng to 100 ng, to induce a range of OspA antibody titers, and exposed to needle challenge with B. burgdorferi s.s. or tick challenge with B. afzelii. Receiver operator characteristics (ROC) curves were constructed for each assay, and the area under the curve (AUC), sensitivity, specificity and Youden Index were calculated. Potential cutoff antibody titers which could be used as correlates of vaccine-induced protection were derived from the maximum Youden Index.

Results

Immunization with OspA-1/2 provided dose-dependent protection against infection with B. burgdorferi s.s. and B. afzelii. Antibody responses detected by all four assays were highly significantly correlated with protection from infection by either B. burgdorferi s.s. (p<0.0001 to 0.0062) or B. afzelii (p<0.0001). ROC analyses of the diagnostic effectiveness of each assay showed the AUC to range between 0.95 and 0.79, demonstrating that all assays distinguish well between infected and non-infected animals. Based on sensitivity, specificity and AUC, the OspA IgG ELISA and SB assays best discriminated between infected and non-infected animals.

Conclusions

All four assays differentiate well between Borrelia-infected and non-infected animals. The relatively simple, high throughput IgG ELISA would be suitable to establish immune correlates of protection for the novel OspA vaccine in clinical trials.