A cultured assay was performed on a select set of positive responders to determine if low-frequency CE-specific responses were present and could be expanded after a 12-day in vitro expansion. This assay's extended period of antigenic stimulation allows for the proliferation of low-frequency HIV-1-specific T cells and therefore achieves a greater sensitivity than does an ex vivo ELISpot assay.33–36 Eighteen participant samples from both treatment groups were cultured with peptides from gag and PRT-RT ARFs but a high background seen in the samples from 7 participants excluded them from further analysis. In the 11 remaining samples with an acceptable background, the magnitude of CE-specific T cells was significantly greater in vaccinees (P < 0.0001; Fig. 4A). Further, CE-specific responses were only detected in the vaccinees and in none of the placebo controls (Fisher exact test, P = 0.06; Fig. 4B). In addition, none of the irrelevant ARF pools (those that were negative at the initial testing) were positive upon culturing. Taken together, these data suggest that CE responses may be induced by this DNA/MVA vaccine regimen, albeit at low frequencies.
In participants of HVTN 205 receiving a vector encoding a noncodon-optimized insert, we were unable to detect significant CE responses ex vivo. Such a finding could be due to a number of factors including the use of cryopreserved specimens and the method of resting cells overnight both of which may have contributed to enhanced cell death. We find these possibilities unlikely because both systems are used extensively by our group and the HVTN and do not significantly impact responses seen to epitopes in the protein reading frame.50–52 It seems more likely that the poor responses observed for CE lies in the fact that even with HIV infection responses to CE are low in magnitude and relatively infrequent.18,19 Further, the average OLP length (15mer, with a range of 8–18mer) exceeded the length of peptides commonly recognized by MHC-I (8–11mer), which potentially reduced the sensitivity to CD8+ T-cell responses in our assays. In addition, the higher frequency of stop codons in ARFs further restricted the size and the number of OLPs that could be artificially synthesized. Accordingly, CE generated through nonconventional translation methods, including stop codon readthrough and doublet decoding, may have been undetectable with the described set of OLPs.32,53 Further, the low immunogenicity of Gag- and PR–RT–CE suggests that MVA/HIV62 may not have been processed efficiently or effectively presented to the immune system.54,55 During infection, translation of PR–RT from the gag–pol transcript relies on a native-1 ribosomal frameshift, which reduces PR–RT expression to one-tenth the level of Gag protein. Because the vector insert maintained the same out-of-frame nucleotide code for the gag and PR–RT regions, vaccine-derived PR–RT levels may have been insufficient for priming central memory T-cell populations and limited the number of effector responses directed against Pol protein in this study. Alternatively, 90% of gag–pol reads do not result in frameshifting, such that translation could occur downstream of gag and generate CE from ARF F2 of PR–RT. Expression from this region is consistent with the trend seen for PR–RT–CE F2 responses (Fig. 3B) and our previous data, which suggested that epitopes in the pol region are predominantly cryptic.18 Nevertheless, a cultured assay was performed with the same OLPs and detected CE-specific T-cell responses exclusively in vaccinees.
Our inability to detect vaccine-induced CE responses ex vivo contrast with what has been reported in rhesus macaques immunized with SIV vaccines.26,56 In fact, CE responses have been reported in up to one-quarter of these vaccinated macaques.57 However, these macaques were vaccinated with SIV rAd5 vectors that potently induce CD8 T-cell responses unlike MVA recombinant vectors, used in our study, that induce relatively weak CD8 responses and are more skewed toward CD4. Additionally, the breadth of vaccine-induced responses is significantly greater in macaques compared with that in humans likely due to the fact that the former primates are able to encode many more MHC type I alleles than are the 6 available to humans.58 As such it seems reasonable to expect that this greater vaccine-induced breadth would extend to CE as well.
Together, our in silico and in vitro analyses suggest that codon optimization negatively impacts the breadth of CE-specific T-cell responses when compared with the repertoire generated through infection or vaccination with a noncodon-optimized vector. Vaccination with the noncodon-optimized MVA/HIV62 vector-induced cellular responses that were low in magnitude and frequency, indicating HIV-1 CE are presented but at levels that may not be adequate for recognition by peripheral T-cells. Still, the immunity afforded to animals vaccinated with naturally encoded constructs supports delivery of noncodon-optimized vectors to expand the pool of vaccine-induced epitopes.59 As future studies determine the importance of CE responses in viral control, our research may provide the direction for developing vaccine designs that increase the breadth of T-cell responses by either preserving as many natural HIV-1 targets as possible or specifically engineering vaccines to express CE as immune targets.
The authors thank all the volunteers who participated in the HVTN 205 study. The authors also thank the HIV Vaccine Trials Network and GeoVax, Inc for their collaboration in supporting this work.
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