Expression of unique chimeric human papilloma virus type 16 (HPV-16) L1-L2 proteins in Pichia pastoris and Hansenula polymorpha.

Cervical cancer is ranked the fourth most common cancer in women worldwide. Despite two prophylactic vaccines being commercially available, they are unaffordable for most women in developing countries. We compared the optimized expression of monomers of the unique HPV type 16 L1-L2 chimeric protein (SAF) in two yeast strains of Pichia pastoris, KM71 (Muts ) and GS115 (Mut+ ), with Hansenula polymorpha NCYC 495 to determine the preferred host in bioreactors. SAF was uniquely created by replacing the h4 helix of the HPV-16 capsid L1 protein with an L2 peptide. Two different feeding strategies in fed-batch cultures of P. pastoris Muts were evaluated: a predetermined feed rate vs. feeding based on the oxygen consumption by maintaining constant dissolved oxygen levels (DO stat). All cultures showed a significant increase in biomass when methanol was fed using the DO stat method. In P. pastoris the SAF concentrations were higher in the Muts strains than in the Mut+ strains. However, H. polymorpha produced the highest level of SAF at 132.10 mg L-1 culture while P. pastoris Muts only produced 23.61 mg L-1 . H. polymorpha showed greater potential for the expression of HPV-16 L1/L2 chimeric proteins despite the track record of P. pastoris as a high-level producer of heterologous proteins.

Intra- and inter-clade cross-reactivity by HIV-1 Gag specific T-cells reveals exclusive and commonly targeted regions: implications for current vaccine trials.

The genetic diversity of HIV-1 across the globe is a major challenge for developing an HIV vaccine. To facilitate immunogen design, it is important to characterize clusters of commonly targeted T-cell epitopes across different HIV clades. To address this, we examined 39 HIV-1 clade C infected individuals for IFN-γ Gag-specific T-cell responses using five sets of overlapping peptides, two sets matching clade C vaccine candidates derived from strains from South Africa and China, and three peptide sets corresponding to consensus clades A, B, and D sequences. The magnitude and breadth of T-cell responses against the two clade C peptide sets did not differ, however clade C peptides were preferentially recognized compared to the other peptide sets. A total of 84 peptides were recognized, of which 19 were exclusively from clade C, 8 exclusively from clade B, one peptide each from A and D and 17 were commonly recognized by clade A, B, C and D. The entropy of the exclusively recognized peptides was significantly higher than that of commonly recognized peptides (p = 0.0128) and the median peptide processing scores were significantly higher for the peptide variants recognized versus those not recognized (p = 0.0001). Consistent with these results, the predicted Major Histocompatibility Complex Class I IC(50) values were significantly lower for the recognized peptide variants compared to those not recognized in the ELISPOT assay (p<0.0001), suggesting that peptide variation between clades, resulting in lack of cross-clade recognition, has been shaped by host immune selection pressure. Overall, our study shows that clade C infected individuals recognize clade C peptides with greater frequency and higher magnitude than other clades, and that a selection of highly conserved epitope regions within Gag are commonly recognized and give rise to cross-clade reactivities.

CTL response to HIV type 1 subtype C is poorly predicted by known epitope motifs.

Cytotoxic T lymphocyte (CTL) responses are thought to be essential for the control of HIV-1 replication in vivo and immunogens that elicit CTL responses are currently a major focus of HIV vaccine research. Here we investigated two aspects of the CTL response to HIV-1 subtype C that are important for vaccine design and efficacy monitoring. First, we assessed the relationship between the CTL response and sequence diversity, using a robust statistical method. While peptides that were most frequently recognized by the CTL response in Nef and p24 tended to be conserved, this was not the case for p17 where epitope recognition coincided with highly variable regions. Second, we investigated the relationship between observed and predicted CTL responses, given the HLA genotype of infected individuals. Only 52% of the Nef peptides and 64% of the Gag peptides that elicited a CTL response contained sequence motifs thought to be required for binding by the HLA-A or -B alleles found in the corresponding patient. In a comparable subtype B dataset a much higher proportion of the peptides that elicited a CTL response were consistent with the patient HLA genotype (96% and 83% for Nef and Gag, respectively). We demonstrate that this difference between subtypes C and B is likely to result from a combination of a tendency for HLA alleles common in Southern African populations to be poorly characterized, as well as a tendency for sequence motifs associated with HLA recognition to be overspecified for sequence variation found in the B clade. Our results suggest that knowledge of HLA binding motifs is likely to be biased toward certain populations and subtypes. This can have important implications for understanding immune escape and predicting vaccine efficacy in the context of populations primarily infected with non-B subtype HIV-1.

Viral dynamics and CD4+ T cell counts in subtype C human immunodeficiency virus type 1-infected individuals from southern Africa.

Defining viral dynamics in natural infection is prognostic of disease progression and could prove to be important for vaccine trial design as viremia may be a likely secondary end point in phase III HIV efficacy trials. There are limited data available on the early course of plasma viral load in subtype C HIV-1 infection in Africa. Plasma viral load and CD4+ T cell counts were monitored in 51 recently infected subjects for 9 months. Individuals were recruited from four southern African countries: Zambia, Malawi, Zimbabwe, and South Africa and the median estimated time from seroconversion was 8.9 months (interquartile range, 5.7-14 months). All were infected with subtype C HIV-1 and median viral loads, measured using branched DNA, ranged from 3.82-4.02 log10 RNA copies/ml from 2-24 months after seroconversion. Viral loads significantly correlated with CD4+ cell counts (r=-0.5, p<0.0001; range, 376-364 cells/mm3) and mathematical modeling defined a median set point of 4.08 log10 (12 143 RNA copies/ml), which was attained approximately 17 months after seroconversion. Comparative measurements using three different viral load platforms (bDNA, Amplicor, and NucliSens) confirmed that viremia in subtype C HIV-1-infected individuals within the first 2 years of infection did not significantly differ from that found in early subtype B infection. In conclusion, the course of plasma viremia, as described in this study, will allow a useful baseline comparator for understanding disease progression in an African setting and may be useful in the design of HIV-1 vaccine trials in southern Africa.

Novel and promiscuous CTL epitopes in conserved regions of Gag targeted by individuals with early subtype C HIV type 1 infection from southern Africa.

Characterization of optimal CTL epitopes in Gag can provide crucial information for evaluation of candidate vaccines in populations at the epicenter of the HIV-1 epidemic. We screened 38 individuals with recent subtype C HIV-1 infection using overlapping consensus C Gag peptides and hypothesized that unique HLA-restricting alleles in the southern African population would determine novel epitope identity. Seventy-four percent of individuals recognized at least one Gag peptide pool. Ten epitopic regions were identified across p17, p24, and p2p7p1p6, and greater than two-thirds of targeted regions were directed at: TGTEELRSLYNTVATLY (p17, 35%); GPKEPFRDYVDRFFKTLRAEQATQDV (p24, 19%); and RGGKLDKWEKIRLRPGGKKHYMLKHL (p17, 15%). After alignment of these epitopic regions with consensus M and a consensus subtype C sequence from the cohort, it was evident that the regions targeted were highly conserved. Fine epitope mapping revealed that five of nine identified optimal Gag epitopes were novel: HLVWASREL, LVWASRELERF, LYNTVATLY, PFRDYVDRFF, and TLRAEQATQD, and were restricted by unique HLA-Cw*08, HLA-A*30/B*57, HLA-A*29/B*44, and HLA-Cw*03 alleles, respectively. Notably, three of the mapped epitopes were restricted by more than one HLA allele. Although these epitopes were novel and restricted by unique HLA, they overlapped or were embedded within previously described CTL epitopes from subtype B HIV-1 infection. These data emphasize the promiscuous nature of epitope binding and support our hypothesis that HLA diversity between populations can shape fine epitope identity, but may not represent a constraint for universal recognition of Gag in highly conserved domains.