Antigen Availability Shapes T Cell Differentiation and Function during Tuberculosis.

CD4 T cells are critical for protective immunity against Mycobacterium tuberculosis (Mtb), the cause of tuberculosis (TB). Yet to date, TB vaccine candidates that boost antigen-specific CD4 T cells have conferred little or no protection. Here we examined CD4 T cell responses to two leading TB vaccine antigens, ESAT-6 and Ag85B, in Mtb-infected mice and in vaccinated humans with and without underlying Mtb infection. In both species, Mtb infection drove ESAT-6-specific T cells to be more differentiated than Ag85B-specific T cells. The ability of each T cell population to control Mtb in the lungs of mice was restricted for opposite reasons: Ag85B-specific T cells were limited by reduced antigen expression during persistent infection, whereas ESAT-6-specific T cells became functionally exhausted due to chronic antigenic stimulation. Our findings suggest that different vaccination strategies will be required to optimize protection mediated by T cells recognizing antigens expressed at distinct stages of Mtb infection.

A Quantitative Analysis of Complexity of Human Pathogen-Specific CD4 T Cell Responses in Healthy M. tuberculosis Infected South Africans.

We performed a quantitative analysis of the HLA restriction, antigen and epitope specificity of human pathogen specific responses in healthy individuals infected with M. tuberculosis (Mtb), in a South African cohort as a test case. The results estimate the breadth of T cell responses for the first time in the context of an infection and human population setting. We determined the epitope repertoire of eleven representative Mtb antigens and a large panel of previously defined Mtb epitopes. We estimated that our analytic methods detected 50-75% of the total response in a cohort of 63 individuals. As expected, responses were highly heterogeneous, with responses to a total of 125 epitopes detected. The 66 top epitopes provided 80% coverage of the responses identified in our study. Using a panel of 48 HLA class II-transfected antigen-presenting cells, we determined HLA class II restrictions for 278 epitope/donor recognition events (36% of the total). The majority of epitopes were restricted by multiple HLA alleles, and 380 different epitope/HLA combinations comprised less than 30% of the estimated Mtb-specific response. Our results underline the complexity of human T cell responses at a population level. Efforts to capture and characterize this broad and highly HLA promiscuous Mtb-specific T cell epitope repertoire will require significant peptide multiplexing efforts. We show that a comprehensive "megapool" of Mtb peptides captured a large fraction of the Mtb-specific T cells and can be used to characterize this response.

T-cell epitope conservation across allergen species is a major determinant of immunogenicity.

BACKGROUND: Patients with pollen allergies are frequently polysensitized. Pollens contain epitopes that are conserved across multiple species. OBJECTIVE: We sought to demonstrate that cross-reactive T cells that recognize conserved epitopes show higher levels of expansion than T cells recognizing monospecific epitopes because of more frequent stimulation. METHOD: RNA was sequenced from 9 pollens, and the reads were assembled de novo into more than 50,000 transcripts. T-cell epitopes from timothy grass (Phleum pratense) were examined for conservation in these transcripts, and this was correlated to their ability to induce T-cell responses. T cells were expanded in vitro with P pratense-derived peptides and tested for cross-reactivity to pollen extracts in ELISpot assays. RESULTS: We found that antigenic proteins are more conserved than nonimmunogenic proteins in P pratense pollen. Additionally, P pratense epitopes that were highly conserved across pollens elicited more T-cell responses in donors with grass allergy than less conserved epitopes. Moreover, conservation of a P pratense peptide at the transcriptomic level correlated with the ability of that peptide to trigger T cells that were cross-reactive with other non-P pratense pollen extracts. CONCLUSION: We found a correlation between conservation of peptides in plant pollens and their T-cell immunogenicity within P pratense, as well as their ability to induce cross-reactive T-cell responses. T cells recognizing conserved epitopes might be more prominent because they can be stimulated by a broader range of pollens and thereby drive polysensitization in allergic donors. We propose that conserved peptides could potentially be used in diagnostic or immunomodulatory approaches that address the issue of polysensitization and target multiple pollen allergies.

Development and validation of a sample sparing strategy for HLA typing utilizing next generation sequencing.

We report the development of a general methodology to genotype HLA class I and class II loci. A Whole Genome Amplification (WGA) step was used as a sample sparing methodology. HLA typing data could be obtained with as few as 300 cells, underlining the usefulness of the methodology for studies for which limited cells are available. The next generation sequencing platform was validated using a panel of cell lines from the International Histocompatibility Working Group (IHWG) for HLA-A, -B, and -C. Concordance with the known, previously determined HLA types was 99%. We next developed a panel of primers to allow HLA typing of alpha and beta chains of the HLA DQ and DP loci and the beta chain of the DRB1 locus. For the beta chain genes, we employed a novel strategy using primers in the intron regions surrounding exon 2, and the introns surrounding exons 3 through 4 (DRB1) or 5 (DQB1 and DPB1). Concordance with previously determined HLA Class II types was also 99%. To increase throughput and decrease cost, we developed strategies combining multiple loci from each donor. Multiplexing of 96 samples per run resulted in increases in throughput of approximately 8-fold. The pipeline developed for this analysis (HLATyphon) is available for download at https://github.com/LJI-Bioinformatics/HLATyphon.

A population response analysis approach to assign class II HLA-epitope restrictions.

Identification of the specific HLA locus and allele presenting an epitope for recognition by specific TCRs (HLA restriction) is necessary to fully characterize the immune response to Ags. Experimental determination of HLA restriction is complex and technically challenging. As an alternative, the restricting HLA locus and allele can be inferred by genetic association, using response data in an HLA-typed population. However, simple odds ratio (OR) calculations can be problematic when dealing with large numbers of subjects and Ags, and because the same epitope can be presented by multiple alleles (epitope promiscuity). In this study, we develop a tool, denominated Restrictor Analysis Tool for Epitopes, to extract inferred restriction from HLA class II-typed epitope responses. This automated method infers HLA class II restriction from large datasets of T cell responses in HLA class II-typed subjects by calculating ORs and relative frequencies from simple data tables. The program is validated by: 1) analyzing data of previously determined HLA restrictions; 2) experimentally determining in selected individuals new HLA restrictions using HLA-transfected cell lines; and 3) predicting HLA restriction of particular peptides and showing that corresponding HLA class II tetramers efficiently bind to epitope-specific T cells. We further design a specific iterative algorithm to account for promiscuous recognition by calculation of OR values for combinations of different HLA molecules while incorporating predicted HLA binding affinity. The Restrictor Analysis Tool for Epitopes program streamlines the prediction of HLA class II restriction across multiple T cell epitopes and HLA types.

Development and validation of a broad scheme for prediction of HLA class II restricted T cell epitopes.

Computational prediction of HLA class II restricted T cell epitopes has great significance in many immunological studies including vaccine discovery. In recent years, prediction of HLA class II binding has improved significantly but a strategy to globally predict the most dominant epitopes has not been rigorously defined. Using human immunogenicity data associated with sets of 15-mer peptides overlapping by 10 residues spanning over 30 different allergens and bacterial antigens, and HLA class II binding prediction tools from the Immune Epitope Database and Analysis Resource (IEDB), we optimized a strategy to predict the top epitopes recognized by human populations. The most effective strategy was to select peptides based on predicted median binding percentiles for a set of seven DRB1 and DRB3/4/5 alleles. These results were validated with predictions on a blind set of 15 new allergens and bacterial antigens. We found that the top 21% predicted peptides (based on the predicted binding to seven DRB1 and DRB3/4/5 alleles) were required to capture 50% of the immune response. This corresponded to an IEDB consensus percentile rank of 20.0, which could be used as a universal prediction threshold. Utilizing actual binding data (as opposed to predicted binding data) did not appreciably change the efficacy of global predictions, suggesting that the imperfect predictive capacity is not due to poor algorithm performance, but intrinsic limitations of HLA class II epitope prediction schema based on HLA binding in genetically diverse human populations.

Sequential Phase 1 and Phase 2 randomized, controlled trials of the safety, immunogenicity and efficacy of combined pre-erythrocytic vaccine antigens RTS,S and TRAP formulated with AS02 Adjuvant System in healthy, malaria naïve adults.

In an attempt to improve the efficacy of the candidate malaria vaccine RTS,S/AS02, two studies were conducted in 1999 in healthy volunteers of RTS,S/AS02 in combination with recombinant Plasmodium falciparum thrombospondin-related anonymous protein (TRAP). In a Phase 1 safety and immunogenicity study, volunteers were randomized to receive TRAP/AS02 (N=10), RTS,S/AS02 (N=10), or RTS,S+TRAP/AS02 (N=20) at 0, 1 and 6-months. In a Phase 2 challenge study, subjects were randomized to receive either RTS,S+TRAP/AS02 (N=25) or TRAP/AS02 (N=10) at 0 and 1-month, or to a challenge control group (N=8). In both studies, the combination vaccine had an acceptable safety profile and was acceptably tolerated. Antigen-specific antibodies, lymphoproliferative responses, and IFN-γ production by ELISPOT assay elicited with the combination vaccine were qualitatively similar to those generated by the single component vaccines. However, post-dose 2 anti-CS antibodies in the RTS,S+TRAP/AS02 vaccine recipients were lower than in the RTS,S/AS02 vaccine recipients. After challenge, 10 of 11 RTS,S+TRAP/AS02 vaccinees, 5 of 5 TRAP/AS02 vaccinees, and 8 of 8 infectivity controls developed parasitemia, with median pre-patent periods of 13.0, 11.0, and 12.0 days, respectively. The absence of any prevention or delay of parasitemia by TRAP/AS02 suggests no apparent added value of TRAP/AS02 as a candidate vaccine. The absence of significant protection or delay of parasitemia in the 11 RTS,S+TRAP/AS02 vaccine recipients contrasts with previous 2 dose studies of RTS,S/AS02. The small sample size did not permit identifying statistically significant differences between the study arms. However, we speculate, within the constraints of the challenge study, that the presence of the TRAP antigen may have interfered with the vaccine efficacy previously observed with this regimen of RTS,S/AS02, and that any future TRAP-based vaccines should consider employing alternative vaccine platforms.

Asymptomatic HLA-A*02:01-restricted epitopes from herpes simplex virus glycoprotein B preferentially recall polyfunctional CD8+ T cells from seropositive asymptomatic individuals and protect HLA transgenic mice against ocular herpes.

Evidence from C57BL/6 mice suggests that CD8(+) T cells, specific to the immunodominant HSV-1 glycoprotein B (gB) H-2(b)-restricted epitope (gB498-505), protect against ocular herpes infection and disease. However, the possible role of CD8(+) T cells, specific to HLA-restricted gB epitopes, in protective immunity seen in HSV-1-seropositive asymptomatic (ASYMP) healthy individuals (who have never had clinical herpes) remains to be determined. In this study, we used multiple prediction algorithms to identify 10 potential HLA-A*02:01-restricted CD8(+) T cell epitopes from the HSV-1 gB amino acid sequence. Six of these epitopes exhibited high-affinity binding to HLA-A*02:01 molecules. In 10 sequentially studied HLA-A*02:01-positive, HSV-1-seropositive ASYMP individuals, the most frequent, robust, and polyfunctional CD8(+) T cell responses, as assessed by a combination of tetramer, IFN-γ-ELISPOT, CFSE proliferation, CD107a/b cytotoxic degranulation, and multiplex cytokine assays, were directed mainly against epitopes gB342-350 and gB561-569. In contrast, in 10 HLA-A*02:01-positive, HSV-1-seropositive symptomatic (SYMP) individuals (with a history of numerous episodes of recurrent clinical herpes disease) frequent, but less robust, CD8(+) T cell responses were directed mainly against nonoverlapping epitopes (gB183-191 and gB441-449). ASYMP individuals had a significantly higher proportion of HSV-gB-specific CD8(+) T cells expressing CD107a/b degranulation marker and producing effector cytokines IL-2, IFN-γ, and TNF-α than did SYMP individuals. Moreover, immunization of a novel herpes-susceptible HLA-A*02:01 transgenic mouse model with ASYMP epitopes, but not with SYMP epitopes, induced strong CD8(+) T cell-dependent protective immunity against ocular herpes infection and disease. These findings should guide the development of a safe and effective T cell-based herpes vaccine.

Previously undescribed grass pollen antigens are the major inducers of T helper 2 cytokine-producing T cells in allergic individuals.

T cells play an important role in the pathogenesis of allergic diseases. However, the proteins considered as potential immunogens of allergenic T-cell responses have traditionally been limited to those that induce IgE responses. Timothy grass (TG) pollen is a well-studied inhaled allergen for which major IgE-reactive allergens have also been shown to trigger T helper 2 (Th2) responses. Here we examined whether other TG pollen proteins are recognized by Th2 responses independently of IgE reactivity. A TG pollen extract was analyzed by 2D gel electrophoresis and IgE/IgG immunoblots using pooled sera from allergic donors. Mass spectrometry of selected protein spots in combination with de novo sequencing of the whole TG pollen transcriptome identified 93 previously undescribed proteins for further study, 64 of which were not targeted by IgE. Predicted MHC binding peptides from the previoulsy undescribed TG proteins were screened for T-cell reactivity in peripheral blood mononuclear cells from allergic donors. Strong IL-5 production was detected in response to peptides from several of the previously undescribed proteins, most of which were not targeted by IgE. Responses against the dominant undescribed epitopes were associated with the memory T-cell subset and could even be detected directly ex vivo after Th2 cell enrichment. These findings demonstrate that a combined unbiased transcriptomic, proteomic, and immunomic approach identifies a greatly broadened repertoire of protein antigens targeted by T cells involved in allergy pathogenesis. The discovery of proteins that induce Th2 cells but are not IgE reactive may allow the development of safer immunotherapeutic strategies.

A strategy to determine HLA class II restriction broadly covering the DR, DP, and DQ allelic variants most commonly expressed in the general population.

Classic ways to determine MHC restriction involve inhibition with locus-specific antibodies and antigen presentation assays with panels of cell lines matched or mismatched at the various loci of interest. However, these determinations are often complicated by T cell epitope degeneracy and promiscuity. We describe a selection of 46 HLA DR, DQ, and DP specificities that provide worldwide population (phenotypic) coverage of almost 90 % at each locus, and account for over 66 % of all genes at each locus. This panel afforded coverage of at least four HLA class II alleles in over 95 % of the individuals in four study populations of diverse ethnicity from the USA and South Africa. Next, a panel of single HLA class II-transfected cell lines, corresponding to these 46 allelic variants was assembled, consisting of lines previously developed and 15 novel lines generated for the present study. The novel lines were validated by assessing their HLA class II expression by FACS analysis, the in vitro peptide binding activity of HLA molecules purified from the cell lines, and their antigen presenting capacity to T cell lines of known restriction. We also show that these HLA class II-transfected cell lines can be used to rapidly and unambiguously determine HLA restriction of epitopes recognized by an individual donor in a single experiment. This panel of lines will enable high throughput determination of HLA restriction, enabling better characterization of HLA class II-restricted T cell responses and facilitating the development of HLA tetrameric staining reagents.

Memory T cells in latent Mycobacterium tuberculosis infection are directed against three antigenic islands and largely contained in a CXCR3+CCR6+ Th1 subset.

An understanding of the immunological footprint of Mycobacterium tuberculosis (MTB) CD4 T cell recognition is still incomplete. Here we report that human Th1 cells specific for MTB are largely contained in a CXCR3(+)CCR6(+) memory subset and highly focused on three broadly immunodominant antigenic islands, all related to bacterial secretion systems. Our results refute the notion that secreted antigens act as a decoy, since both secreted proteins and proteins comprising the secretion system itself are targeted by a fully functional T cell response. In addition, several novel T cell antigens were identified which can be of potential diagnostic use, or as vaccine antigens. These results underline the power of a truly unbiased, genome-wide, analysis of CD4 MTB recognition based on the combined use of epitope predictions, high throughput ELISPOT, and T cell libraries using PBMCs from individuals latently infected with MTB.

T cell responses to known allergen proteins are differently polarized and account for a variable fraction of total response to allergen extracts.

A panel of 133 allergens derived from 28 different sources, including fungi, trees, grasses, weeds, and indoor allergens, was surveyed utilizing prediction of HLA class II-binding peptides and ELISPOT assays with PBMC from allergic donors, resulting in the identification of 257 T cell epitopes. More than 90% of the epitopes were novel, and for 14 allergen sources were the first ever identified to our knowledge. The epitopes identified in the different allergen sources summed up to a variable fraction of the total extract response. In cases of allergens in which the identified T cell epitopes accounted for a minor fraction of the extract response, fewer known protein sequences were available, suggesting that for low epitope coverage allergen sources, additional allergen proteins remain to be identified. IL-5 and IFN-γ responses were measured as prototype Th2 and Th1 responses, respectively. Whereas in some cases (e.g., orchard grass, Alternaria, cypress, and Russian thistle) IL-5 production greatly exceeded IFN-γ, in others (e.g., Aspergillus, Penicillum, and alder) the production of IFN-γ exceeded IL-5. Thus, different allergen sources are associated with variable polarization of the responding T cells. The present study represents the most comprehensive survey to date of human allergen-derived T cell epitopes. These epitopes might be used to characterize T cell phenotype/T cell plasticity as a function of seasonality, or as a result of specific immunotherapy treatment or varying disease severity (asthma or rhinitis).

Dissecting mechanisms of immunodominance to the common tuberculosis antigens ESAT-6, CFP10, Rv2031c (hspX), Rv2654c (TB7.7), and Rv1038c (EsxJ).

Diagnosis of tuberculosis often relies on the ex vivo IFN-γ release assays QuantiFERON-TB Gold In-Tube and T-SPOT.TB. However, understanding of the immunological mechanisms underlying their diagnostic use is still incomplete. Accordingly, we investigated T cell responses for the TB Ags included in the these assays and other commonly studied Ags: early secreted antigenic target 6 kDa, culture filtrate protein 10 kDa, Rv2031c, Rv2654c, and Rv1038c. PBMC from latently infected individuals were tested in ex vivo ELISPOT assays with overlapping peptides spanning the entirety of these Ags. We found striking variations in prevalence and magnitude of ex vivo reactivity, with culture filtrate protein 10 kDa being most dominant, followed by early secreted antigenic target 6 kDa and Rv2654c being virtually inactive. Rv2031c and Rv1038c were associated with intermediate patterns of reactivity. Further studies showed that low reactivity was not due to lack of HLA binding peptides, and high reactivity was associated with recognition of a few discrete dominant antigenic regions. Different donors recognized the same core sequence in a given epitope. In some cases, the identified epitopes were restricted by a single specific common HLA molecule (selective restriction), whereas in other cases, promiscuous restriction of the same epitope by multiple HLA molecules was apparent. Definition of the specific restricting HLA allowed to produce tetrameric reagents and showed that epitope-specific T cells recognizing either selectively or promiscuously restricted epitopes were predominantly T effector memory. In conclusion, these results highlight the feasibility of more clearly defined TB diagnostic reagent.

Molecular determinants of T cell epitope recognition to the common Timothy grass allergen.

We investigated the molecular determinants of allergen-derived T cell epitopes in humans utilizing the Phleum pratense (Timothy grass) allergens (Phl p). PBMCs from allergic individuals were tested in ELISPOT assays with overlapping peptides spanning known Phl p allergens. A total of 43 distinct antigenic regions were recognized, illustrating the large breadth of grass-specific T cell epitopes. Th2 cytokines (as represented by IL-5) were predominant, whereas IFN-gamma, IL-10, and IL-17 were detected less frequently. Responses from specific immunotherapy treatment individuals were weaker and less consistent, yet similar in epitope specificity and cytokine pattern to allergic donors, whereas nonallergic individuals were essentially nonreactive. Despite the large breadth of recognition, nine dominant antigenic regions were defined, each recognized by multiple donors, accounting for 51% of the total response. Multiple HLA molecules and loci restricted the dominant regions, and the immunodominant epitopes could be predicted using bioinformatic algorithms specific for 23 common HLA-DR, DP, and DQ molecules. Immunodominance was also apparent at the Phl p Ag level. It was found that 52, 19, and 14% of the total response was directed to Phl p 5, 1, and 3, respectively. Interestingly, little or no correlation between Phl p-specific IgE levels and T cell responses was found. Thus, certain intrinsic features of the allergen protein might influence immunogenicity at the level of T cell reactivity. Consistent with this notion, different Phl p Ags were associated with distinct patterns of IL-5, IFN-gamma, IL-10, and IL-17 production.

A novel HIV T helper epitope-based vaccine elicits cytokine-secreting HIV-specific CD4+ T cells in a Phase I clinical trial in HIV-uninfected adults.

A Phase I human vaccine trial of a novel polypeptide vaccine of HIV T helper epitopes (EP-1043) and a DNA vaccine of HIV CTL epitopes was conducted in 84 healthy adult volunteers. The vaccine immunogenicity was assessed by an intracellular cytokine staining assay for IL-2, IL-4, TNF-alpha and IFN-gamma. Sixty eight percent (32/47) of subjects had a positive CD4+ T response after receiving two vaccinations of the polypeptide vaccine. The responding CD4+ T cells made various combinations of IL-2, IL-4, IFN-gamma, and TNF-alpha. The study demonstrated that the EP-1043 vaccine is safe, well-tolerated, and immunogenic.

Blood stage malaria vaccine eliciting high antigen-specific antibody concentrations confers no protection to young children in Western Kenya.

OBJECTIVE: The antigen, falciparum malaria protein 1 (FMP1), represents the 42-kDa C-terminal fragment of merozoite surface protein-1 (MSP-1) of the 3D7 clone of P. falciparum. Formulated with AS02 (a proprietary Adjuvant System), it constitutes the FMP1/AS02 candidate malaria vaccine. We evaluated this vaccine's safety, immunogenicity, and efficacy in African children. METHODS: A randomised, double-blind, Phase IIb, comparator-controlled trial.The trial was conducted in 13 field stations of one mile radii within Kombewa Division, Nyanza Province, Western Kenya, an area of holoendemic transmission of P. falciparum. We enrolled 400 children aged 12-47 months in general good health.Children were randomised in a 1ratio1 fashion to receive either FMP1/AS02 (50 microg) or Rabipur(R) rabies vaccine. Vaccinations were administered on a 0, 1, and 2 month schedule. The primary study endpoint was time to first clinical episode of P. falciparum malaria (temperature >/=37.5 degrees C with asexual parasitaemia of >/=50,000 parasites/microL of blood) occurring between 14 days and six months after a third dose. Case detection was both active and passive. Safety and immunogenicity were evaluated for eight months after first immunisations; vaccine efficacy (VE) was measured over a six-month period following third vaccinations. RESULTS: 374 of 400 children received all three doses and completed six months of follow-up. FMP1/AS02 had a good safety profile and was well-tolerated but more reactogenic than the comparator. Geometric mean anti-MSP-1(42) antibody concentrations increased from1.3 microg/mL to 27.3 microg/mL in the FMP1/AS02 recipients, but were unchanged in controls. 97 children in the FMP1/AS02 group and 98 controls had a primary endpoint episode. Overall VE was 5.1% (95% CI: -26% to +28%; p-value = 0.7). CONCLUSIONS: FMP1/AS02 is not a promising candidate for further development as a monovalent malaria vaccine. Future MSP-1(42) vaccine development should focus on other formulations and antigen constructs. TRIAL REGISTRATION: Clinicaltrials.gov NCT00223990.

Rational design of a multiepitope vaccine encoding T-lymphocyte epitopes for treatment of chronic hepatitis B virus infections.

Protein sequences from multiple hepatitis B virus (HBV) isolates were analyzed for the presence of amino acid motifs characteristic of cytotoxic T-lymphocyte (CTL) and helper T-lymphocyte (HTL) epitopes with the goal of identifying conserved epitopes suitable for use in a therapeutic vaccine. Specifically, sequences bearing HLA-A1, -A2, -A3, -A24, -B7, and -DR supertype binding motifs were identified, synthesized as peptides, and tested for binding to soluble HLA. The immunogenicity of peptides that bound with moderate to high affinity subsequently was assessed using HLA transgenic mice (CTL) and HLA cross-reacting H-2(bxd) (BALB/c x C57BL/6J) mice (HTL). Through this process, 30 CTL and 16 HTL epitopes were selected as a set that would be the most useful for vaccine design, based on epitope conservation among HBV sequences and HLA-based predicted population coverage in diverse ethnic groups. A plasmid DNA-based vaccine encoding the epitopes as a single gene product, with each epitope separated by spacer residues to enhance appropriate epitope processing, was designed. Immunogenicity testing in mice demonstrated the induction of multiple CTL and HTL responses. Furthermore, as a complementary approach, mass spectrometry allowed the identification of correctly processed and major histocompatibility complex-presented epitopes from human cells transfected with the DNA plasmid. A heterologous prime-boost immunization with the plasmid DNA and a recombinant MVA gave further enhancement of the immune responses. Thus, a multiepitope therapeutic vaccine candidate capable of stimulating those cellular immune responses thought to be essential for controlling and clearing HBV infection was successfully designed and evaluated in vitro and in HLA transgenic mice.

A phase I/IIa safety, immunogenicity, and efficacy bridging randomized study of a two-dose regimen of liquid and lyophilized formulations of the candidate malaria vaccine RTS,S/AS02A in malaria-naïve adults.

We conducted an open-label safety and immunogenicity bridging study that compared liquid and lyophilized formulations of the candidate malaria vaccine RTS,S formulated in AS02A in 34 healthy, malaria-naïve adults at WRAIR. Volunteers received two doses of either formulation on a 0, 1-month schedule. Both vaccines were well tolerated and similarly immunogenic. Nineteen of 25 subjects who received the lyophilized formulation and six infectivity controls underwent sporozoite challenge to assess vaccine efficacy. All six controls had parasitemia detectable by thick blood smear by day 13 (mean pre-patent period 12.3 days; range 11-13). In the vaccine group, 8 of 19 vaccinees did not develop malaria and were completely protected (i.e., 42%). Among the 11 vaccinees who did become infected, the mean pre-patent period was delayed (14.4 days; range 13-18). The two formulations of RTS,S were equally safe and immunogenic, and the lyophilized formulation showed similar levels of efficacy against sporozoite challenge to that conferred by the liquid formulation in previous studies.

Absence of viral escape within a frequently recognized HLA-A26-restricted CD8+ T-cell epitope targeting the functionally constrained hepatitis C virus NS5A/5B cleavage site.

CD8(+) T-cell responses are central for the resolution of hepatitis C virus (HCV) infection, and viral escape from these CD8(+) T-cell responses has been suggested to play a major role in HCV persistence. However, the factors determining the emergence of CD8 escape mutations are not well understood. Here, the first identification of four HLA-A26-restricted CD8(+) T-cell epitopes is reported. Of note, two of these four epitopes are located in the NS3/4A and NS5A/5B cleavage sites. The latter epitope is targeted in all (three of three) patients with acute, resolving HCV infection and in a relatively high proportion (four of 14) of patients with chronic HCV infection. Importantly, the epitope corresponding to the NS5A/5B cleavage site is characterized by the complete absence of sequence variations, despite the presence of functional virus-specific CD8(+) T cells in our cohort. These results support previous findings that showed defined functional constraints within this region. They also suggest that the absence of viral escape may be determined by viral fitness cost and highlight an attractive target for immunotherapies.