ABSTRACT
Prime-boost vaccination strategies against HIV-1 often include multiple variants for a given immunogen for better coverage of the extensive viral diversity. To study the immunologic effects of this approach, we characterized breadth, phenotype, function, and specificity of Gag-specific T cells induced by a DNA-prime modified vaccinia virus Ankara (MVA)-boost vaccination strategy, which uses mismatched Gag immunogens in the TamoVac 01 phase IIa trial. Healthy Tanzanian volunteers received three injections of the DNA-SMI vaccine encoding a subtype B and AB-recombinant Gagp37 and two vaccinations with MVA-CMDR encoding subtype A Gagp55. Gag-specific T-cell responses were studied in 42 vaccinees using fresh peripheral blood mononuclear cells. After the first MVA-CMDR boost, vaccine-induced gamma interferon-positive (IFN-γ+) Gag-specific T-cell responses were dominated by CD4+ T cells (P < 0.001 compared to CD8+ T cells) that coexpressed interleukin-2 (IL-2) (66.4%) and/or tumor necrosis factor alpha (TNF-α) (63.7%). A median of 3 antigenic regions were targeted with a higher-magnitude median response to Gagp24 regions, more conserved between prime and boost, compared to those of regions within Gagp15 (not primed) and Gagp17 (less conserved; P < 0.0001 for both). Four regions within Gagp24 each were targeted by 45% to 74% of vaccinees upon restimulation with DNA-SMI-Gag matched peptides. The response rate to individual antigenic regions correlated with the sequence homology between the MVA- and DNA Gag-encoded immunogens (P = 0.04, r2 = 0.47). In summary, after the first MVA-CMDR boost, the sequence-mismatched DNA-prime MVA-boost vaccine strategy induced a Gag-specific T-cell response that was dominated by polyfunctional CD4+ T cells and that targeted multiple antigenic regions within the conserved Gagp24 protein.
IMPORTANCE Genetic diversity is a major challenge for the design of vaccines against variable viruses. While including multiple variants for a given immunogen in prime-boost vaccination strategies is one approach that aims to improve coverage for global virus variants, the immunologic consequences of this strategy have been poorly defined so far. It is unclear whether inclusion of multiple variants in prime-boost vaccination strategies improves recognition of variant viruses by T cells and by which mechanisms this would be achieved, either by improved cross-recognition of multiple variants for a given antigenic region or through preferential targeting of antigenic regions more conserved between prime and boost. Engineering vaccines to induce adaptive immune responses that preferentially target conserved antigenic regions of viral vulnerability might facilitate better immune control after preventive and therapeutic vaccination for HIV and for other variable viruses.
INTRODUCTION
High antigenic variability of common viruses causing either chronic (e.g., human immunodeficiency virus [HIV] and hepatitis C virus [HCV]) or acute (e.g., influenza virus and dengue virus) disease complicates the design of efficacious vaccines. Vaccines against such variable viruses optimally should induce adaptive immune responses that target all variants of a vulnerable antigenic region to prevent infection or, if that cannot be achieved, at least facilitate immune control of viral replication to prevent disease progression. All virus proteomes, even the most variable ones, contain conserved regions, where functional constraints limit extensive sequence variability. Designing vaccines to focus immune recognition toward such conserved regions could be a viable strategy to improve vaccine efficacy against variable pathogens (1, 2).
HIV is a good example of a highly variable virus. It causes persistent infection and rapidly escapes the HIV envelope (Env)-specific antibody response. Extensive sequence variability and glycosylation of the Env protein complicate Env-based vaccine design (3, 4). In contrast, sequence variability within the group-specific antigen (Gag) is more limited due to functional constraints but still differs between the capsid protein p24, the matrix protein p17, and proteins of the p15-encoding region (nucleocapsid and virion assembly proteins). An extensive body of evidence supports the concept that vaccine induction of Gag-specific T-cell responses could be beneficial; Gag-specific T-cell responses are linked to viral control during chronic HIV infection (5–10). The breadth of Gag recognition by CD8+ T cells is associated with better viral control and slower disease progression (9, 10). Furthermore, the protective mechanism mediated by HLA class I alleles has been linked to CD8+ T-cell recognition of defined Gag regions (10, 11). Similarly, Gag-specific CD4+ T-cell responses appear to contribute to viral control, sharing similar features with the CD8+ T-cell response (12–14). In rhesus monkeys, vaccine-induced Gag-specific T-cell responses correlate with postchallenge immune control and prolonged survival after SIV challenge (15). Together, these data support the concept that induction of strong and broad Gag-specific T-cell responses targeting common sequence variants could improve HIV vaccine efficacy against diverse HIV variants.
The multisubtype TaMoVac DNA/MVA regimen used in the TaMoVac 01 phase IIa trial induced strong Gag-specific T-cell responses (16, 17). This offered the opportunity to address the hypothesis that delivery of nonidentical but related immunogens preferentially induces T-cell responses to antigenic regions more conserved between the immunogens. We found that the sequence-mismatched TaMoVac01 DNA-MVA vaccination induced broad recognition of conserved antigenic regions within Gagp24.
RESULTS
Relative conservation of Gag p24 in a mixed-subtype epidemic.In order to define the degree of conservation within Mbeyan HIV Gag sequences, we analyzed previously published sequences and determined the distribution of subtypes and recombinant forms (RF). Pure subtype C sequences were found most frequently (57.1%), followed by unique subtype A-containing recombinant forms (RFs) (20.9%), pure subtype A Gag sequences (19.8%), and CD RFs (2.2%, Fig. 1). In order to estimate the variability at each Gag amino acid position, the Shannon entropy score was calculated (Fig. 1B). Gag p24 showed the highest conservation, with 82.6% of amino acid positions with an entropy score below 0.5 compared to the less conserved p17 (55.7%) and the least conserved p15 (44.8%). Similarly, the median Shannon entropy score was lowest for p24 (0.14), followed by p17 (0.37) and p15 (0.49).
Diversity of HIV-1 Gag protein sequences originating from the Mbeya region. (A) The distribution of subtypes and unique recombinant forms of Gag polyprotein sequences from 91 HIV-infected subjects from the Mbeya region is shown in the pie chart. (B) Shannon entropy plot generated from these Gag sequences is shown.
Preferential induction of T cells targeting Gagp24 protein by sequence-mismatched DNA/MVA vaccination.The DNA vaccine included two plasmids encoding SMI-Gagp37 with an identical p17 subtype B sequence linked to subtype B or subtype A p24 sequences (Fig. 2). The MVA-CMDR-Gagp55 boost encoded a subtype A Gagp55 and included the p15 region. Within the p17 region, 21.2% (28 of 132) of amino acid positions were mismatched between the DNA-SMI prime and MVA-CMDR boost. The Gagp24 subtype A and subtype B sequences included in the DNA-SMI prime differed from the MVA-CMDR boost in 7.8% (18 mismatches in 231) and 11.7% (27 mismatches in 231) of amino acid positions, respectively. The p15 region represented a 100% mismatch to the DNA-SMI prime.
Immunogen sequences included in the DNA-Gag prime and modified vaccinia virus Ankara (MVA)-Gag boost and their coverage by peptide pools. The seven DNA peptide pools covered the Gagp37 region consisting of p17 (blue) and p24 (gray), whereas the nine MVA-Gag peptide pools covered the Gagp55 precursor protein, including the p15 region (red) in addition to p17 and p24. The p15 region was only covered by MVA-Gag peptide pools 8 and 9.
IFN-γ+ Gag-specific T-cell responses were not detected during the prevaccination visit upon MVA-CMDR-Gagp55 restimulation but were present in 15% of vaccinees upon DNA-SMI-Gagp37 restimulation (range, 60 to 165 spot-forming cells [SFC]/106 peripheral blood mononuclear cells [PBMC]; data not shown). Gag-specific T-cell numbers already peaked after the first MVA-CMDR boost, with a median of 228 SFC/106 PBMC and significantly lower numbers of Gag-specific T cells after the second boost (P < 0.05 for both peptide pools). Magnitude of T-cell responses against the control peptide pools CEF (median, 83 and 90 SFC/106 PBMC; P = 0.58) and CMVpp65 (median, 1,243 and 938 SFC/106 PBMC; P = 0.2) were comparable for both visits.
Because vaccine-induced IFN-γ+ Gag-specific T-cell responses peaked after the first MVA-CMDR boost, we focus on this time point in subsequent analyses. Intracellular cytokine staining (ICS) data from 41 vaccine recipients were eligible for further analyses. IFN-γ+ Gag-specific CD4 and CD8+ T-cell responses were detected in 30 and 14 of 41 subjects, respectively, after restimulation with the MVA-CMDR-Gagp55 peptide pool (Fisher's exact test; P = 0.0008). Likewise, significantly higher frequencies of CD4+ than CD8+ IFN-γ+ Gag-specific T cells were detected after restimulation with either the MVA-CMDR-Gagp55 (median, 0.04% versus 0.01%; P < 0.0001) (Fig. 3A) or the DNA-SMI-Gagp37 (median, 0.06% versus 0.01%; P = 0.0002) peptide pool after the first MVA-CMDR boost. IFN-γ+ Gag-specific CD4 T cells frequently coexpressed interleukin-2 (IL-2) (mean of both Gag peptide pools, 66.4%) or tumor necrosis factor alpha (TNF-α) (63.7%), with 49.9% coexpressing IL-2 and TNF-α and high concordance between the two tested Gag peptide pools (Fig. 3B). The CCR5 ligand Mip-1β and degranulation marker CD107 were coexpressed by only 20% (CMDR-Gagp55, 13%) and 11% (CMDR-Gagp55, 7%) of IFN-γ+ Gag-specific CD4+ T cells, respectively. In contrast, high frequencies of IFN-γ+ CD4+ T cells coexpressed Mip-1β (59%) and/or CD107 (53%) after restimulation with CMVpp65 peptides, including a significant proportion (16%) of polyfunctional cells expressing all assessed functional markers, which is concordant with previous reports (18). Almost 75% of IFN-γ+ Gag-specific CD8 T cells coexpressed Mip-1β, and almost 50% were CD107+, indicating degranulation of cytotoxic granules.
Phenotype, function, and breadth of vaccine-induced Gag-specific T-cell responses. (A) Representative dot plots for the analyses of Gag-specific CD4 and CD8 T-cell functions are shown. (B) Frequencies of IFN-γ+, CD4+, and CD8+ T cells after stimulation of freshly isolated PBMC with whole MVA-CMDR-Gagp55 (left) or DNA-SMI-Gagp37 (right) peptide pools in 41 vaccinees are shown. (C) Coexpression of additional functions (TNF-α, Mip-1β, IL-2, and the degranulation marker CD107) for IFN-γ+ CD4+ (left) and IFN-γ+ CD8+ (right) T cells. The four color-coded arcs indicate the proportion of cells coexpressing the four additional functions. The color-coded pies symbolize the 16 possible functional combinations. Intracellular cytokine staining was performed using fresh PBMC stimulated overnight with the indicated antigens as well as the control antigens Staphylococcus enterotoxin B and CMVpp65. (D) The number of different antigenic regions (linear peptide pools, x axis) recognized by TaMoVac vaccinees (n = 42) is shown and was determined using the IFN-γ ELISPOT assay. The frequency of subjects with a given Gag response breadth is indicated on the y axis. Nine and 7 linear peptide pools matching MVA-CMDR-Gagp55 (gray bars) or DNA-SMI-Gagp37 (black bars), respectively, subdivided Gag into distinct antigenic regions. Statistical comparison for panel B was performed using Wilcoxon matched-pairs signed-rank test.
Breadth and specificity of Gag-specific T-cell recognition were determined in 42 vaccine recipients using the IFN-γ ELISPOT assay on freshly isolated PBMC after restimulation with linear consecutive peptide pools matching for MVA-CMDR-Gagp55 (n = 9 pools) and DNA-SMI-Gagp37 (n = 7 pools). The median breadth of Gag recognition was 3 peptide pools (range, 0 to 8) (Fig. 3C). The majority of the 42 vaccine recipients responded to at least one DNA-SMI-Gagp37 (n = 36; 85.7%) or MVA-CMDR-Gagp55 (n = 33; 78.6%) peptide pool. In order to compare the magnitude of T-cell responses against the different Gag regions and account for sequence lengths of p17, p24, and p15, we calculated average SFC values per 15mer peptide for each Gag region. Antigenic regions within p24 were recognized at a higher median magnitude (14.55 SFC/106 PBMC/15mer peptide) compared to those in the more variable p17 and p15 regions (both 6.52 SFC/106 PBMC/15mer peptide; P < 0.0001) (Fig. 4A). The magnitude of response to p17 (median of 6.52 SFC/106 PBMC/15mer peptide for MVA-CMDR and 6.0 SFC/106 PBMC/15mer_peptide for DNA-SMI) was similar to the response to p15 (P = 0.95).
Recognition of Gag antigenic regions by vaccine-induced T cells. (A) A comparison of the magnitude of Gag-specific T-cell responses (y axis) targeting antigenic regions within p17, p24, and p15 normalized per 15mer peptide is shown. (B) The frequency of responders is shown for different Gag regions. Vaccine-induced T-cell responses were characterized using IFN-γ ELISPOT assay in 42 participants after stimulation of fresh PBMC with 9 and 7 peptide pools matching MVA-CMDR-Gagp55 (gray bars) and DNA-Gagp37 (black bars), respectively. (C) The frequency of responders for individual MVA-CMDR-Gagp55 matched peptides is shown and is based on 23 subjects with at least 1 detectable response against an individual peptide during fine mapping using cryopreserved instead of fresh PBMC. (D) The key data for the most frequently recognized peptides are shown. The cutoff for a positive response was 2-fold above the level of the unstimulated control. Corresponding Gag regions p15, p17, and p24 are indicated in panels B and C.
Ten of the 42 vaccine recipients (23.8%) mounted SMI Gagp17-specific T-cell responses, and 36 volunteers (85.7%) responded to SMI-Gagp24. Eight vaccine recipients (19.0%) responded to CMDR-Gagp17, 32 (76.2%) to CMDR-Gagp24, and 8 (19.0%) to CMDR-Gagp15. Recognition of individual antigenic regions within Gagp24 was comparable between DNA and MVA variants (Fig. 4B). Pools 5 (HxB amino acids [aa] 225 to 290) and 6 (aa 275 to 347) were immunodominant, with 57.1% (CMDR-Gag peptides, 47.6%) and 73.8% (CMDR-Gag peptides, 57.1%) of responders after restimulation with the SMI Gag peptides, respectively. Pool 5 was targeted with a median magnitude of 38.65 SFC/106 PBMC/15mer (SMI Gag peptides) and 35.19 SFC/106 PBMC/15mer (CMDR-Gag peptides). Pool 6 was targeted with a median magnitude of 27.74 SFC/106 PBMC/15mer (SMI-Gag peptides) and 29.79 SFC/106 PBMC/15mer (CMDR-Gag peptides).
We next mapped individual MVA-CMDR-Gagp55 peptide responses (Fig. 4C) in 39 vaccine recipients using cryopreserved PBMC. Results from 3 vaccine recipients were excluded because of invalid ELISPOT assay results. Positive peptide-specific ELISPOT assay responses were detected in 23 of 39 (60%) vaccine recipients. The 23 responders almost exclusively recognized peptides located within the p24 region. Four individual peptide responses were detected in p17 and two within p15. When counting responses to consecutive peptides as a single antigenic region, these 23 responders recognized a mean of 4 antigenic regions, ranging from 1 (26% of subjects [6/23]) to 12 (4.3% [1/23]) antigenic regions recognized. The peptide YVDRFYKTLRAEQAT (pool 6, HxB position aa 296 to 310) was the most frequently recognized, with 39.13% (9 of 23) being responders, followed by the peptide YKRWIILGLNKIVRMY (pool 5, aa 262 to 277, 30.43% responders). Four peptides within p24 were recognized with an equal frequency of 26.09%: GATPQDLNMMLNIVGG (pool 4, aa 178 to 193), IAGTTSTLQEQIGWMT (pool 5, aa 236 to 251), ILGLNKIVRMYSPVSI (9) (pool 5, aa 267 to 282), and WMTETLLVQNANPDCK (pool 6, aa 316 to 331). As shown in Fig. 4D, the six most frequently recognized antigenic regions had a 0- or 1-aa difference between the SMI-DNA prime Gagp37 sequences (DNA B or BA) and the MVA-CMDR boost Gagp55 sequences. These data indicate that hotspots of the vaccine-induced Gag recognition by T cells were located exclusively within p24, whereas recognition of antigenic regions within p17 and p15 appeared attenuated.
We next compared the frequency of responders to a given antigenic region with the region-specific amino acid mismatches between the DNA- and the MVA-encoded Gag sequences using linear regression analysis (Fig. 5). Plotting the frequency of responders against the sequence heterogeneity between the encoded amino acid sequence of the DNA-SMI-Gagp37 prime and the MVA-CMDR-Gagp55 boost for each linear peptide pool, we found a linear correlation between region-specific amino acid mismatches and the frequency of responders to a given antigenic region (r2 = 0.69, P = 0.04 [DNA-SMI-Gagp37] and r2 = 0.45, P = 0.07 [MVA-CMDR-Gagp55]) (Fig. 5A), suggesting that higher levels of sequence conservation between DNA-SMI-Gagp37 prime and MVA-CMDR-Gagp55 boost contribute to higher frequencies of recognition within more conserved Gag regions.
Linear regression analyses between DNA-SMI-Gagp37 and MVA-CMDR-Gagp55 sequence mismatches and induced T-cell responses targeting specific antigenic regions. (A) A linear regression analysis was performed to study the association of immunogen-located amino acid mismatches and the respective T-cell response rate to the DNA-SMI-Gagp37 pools 1 to 6. T-cell responses were detected using the IFN-γ ELISPOT assay, and freshly isolated PBMC pool 7 was excluded because it contains only 3 instead of 11 peptides. A comparison of mismatches between MVA-CMDR-Gagp55 and the DNA-SMI-Gagp37B sequence and the respective T-cell response rate (B) or the magnitude of responses (C) for single-peptide-specific T-cell responses was detected using the IFN-γ ELISPOT assay and cryopreserved PBMC.
The link between the level of sequence conservation within a given antigenic region and its recognition could be substantiated further by linear regression analysis. The number of amino acid mismatches between priming DNA Gagp24-encoded subtype B sequence and boosting Gag amino acid sequences (MVA) correlated with the frequency of responders (r2 = 0.01 and P = 0.0332) (Fig. 5B) and the magnitude of response (P = 0.04, r2 = 0.04) (Fig. 5C) to a given Gagp24 peptide. These data support the concept that the heterologous sequence prime-boost vaccination strategy applied during TaMoVac 01 contributed to preferential recognition of the more conserved antigenic regions within Gag.
DISCUSSION
Genetic diversity poses a major challenge to the design of efficacious vaccines against HIV-1 and other variable viruses. Several vaccination strategies have been tested to address this extensive diversity of HIV-1 (2). The TaMoVac 01 study incorporated multiple variants of the same immunogen in the vaccine formulation (16, 17, 19). The immunologic consequences of such vaccination strategies incorporating multiple variants and, in particular, the impact on the pattern of antigen recognition have been defined only poorly so far. Inclusion of three immunogen variants elicited strong Gag-specific T-cell responses in the TaMoVac 01 and HIVIS studies (16, 17), providing the opportunity to study parameters that potentially influence the immunodominance pattern of vaccine-induced Gag recognition by T cells. The analysis of 91 Gag sequences confirmed previous reports that the capsid antigen p24 shows the highest degree of conservation, followed by the less conserved p17 and p15 (20).
In TaMoVac 01 vaccinees, antigenic regions within conserved Gagp24 regions were preferentially targeted after the first MVA-CMDR boost. A comparative analysis of HIV-1 recombinant Ad-5 T-cell-based vaccine clinical trials Merck16, HVTN 054, and HVTN 502/Step, which included closely related Gag immunogen sequences and did not include DNA priming, found considerable variation in the recognition of Gag regions (21). HVTN 054 showed an accumulation of hotspots within Gagp24, whereas hotspots of T-cell recognition were more evenly distributed within Gag for Merck16 and HVTN 502/Step. In comparison, the sequence-mismatched DNA/MVA TaMoVac vaccine focused T-cell responses even more on highly conserved Gagp24 regions. The strength of induced T-cell responses against individual antigenic regions in Gagp24 correlated with the degree of conservation between the SMI-DNA-encoded subtype B and MVA-CMDR-encoded subtype A sequence variants. While no such correlation was detected for the DNA-encoded subtype A variant, the six most frequently targeted peptides showed none or one amino acid mismatch when taking both DNA-encoded Gagp24 variants into account. Hence, immune recognition of peptides with a high degree of variability between MVA-CMDR and DNA-Gagp37.1 (Fig. 2, subtype A) was probably primed by the other DNA-Gagp37 variant (subtype B).
What is the mechanism underlying this preferential targeting of more conserved antigenic regions within Gagp24 in TaMoVac01 vaccinees? One possibility is that induction of T-cell responses toward more variable epitopes, which are more common in Gagp17 and Gagp15, was negatively affected by increasing numbers of mismatched amino acid positions within a given epitope. Immunodominant Gag-specific CD8+ T-cell populations targeting the epitope variants TL9M7 (TPQDLNMML) and TL9T7 (TPQDLNTML) during natural HIV subtype A and subtype C infections are completely different in their clonotypic composition (22). It is therefore plausible that certain T-cell clonotypes induced by one immunogen sequence cannot be boosted by certain other sequence variants or that boosting is suboptimal; however, if T-cell clonotypes partially overlap in their recognition of the two epitope variants, these cross-reactive clonotypes would be boosted more strongly, resulting in improved variant cross-recognition and focusing toward such cross-reactive clonotypes. Our data show that recognition of epitopes with more than 2 mismatches is completely abrogated (Fig. 5C and D). Therefore, a high number of epitope variant mismatches is likely to abolish any efficient boosting. Of note, based on our analyses of 91 Mbeyan sequences, the tested CMDR-Gag sequence variants for the peptides P1 to P6 always closely matched the most frequent Mbeyan variants with ≤2 amino acid substitutions. In summary, a variety of mechanisms probably contributed to preferential T-cell recognition of more conserved Gag regions in the TaMoVac01 study using mismatched immunogen sequences.
All immunodominant regions recognized by the TaMoVac 01 vaccinees have also been identified in previous studies. P1 (YVDRFYKTLRAEQAT) is an immunodominant target for CD4+ and CD8+ T cells during early and chronic infection (14, 23, 24) and was also a hot spot in the HVTN502/Step and HVTN 054 trials (21). P2 (YKRWIILGLNKIVRMY) is frequently targeted by CD4+ T cells during early and chronic HIV infection (14) and was a hot spot in the Step trial (21). P3 (GATPQDLNMMLNIVGG) contains the highly immunodominant B42/B81-restricted TL9 epitope (22, 24–26). P4 to P6 also contained previously described epitopes (14, 23). Inherent immunogenicity of these peptide sequences, P1 to P6, also could contribute to their preferential recognition, as these were also frequently recognized by T cells in natural infection or in other trials.
Most individual peptide-specific T-cell responses after the first MVA-CMDR boost were likely mediated by CD4+ rather than CD8+ T cells, even though we were not able to phenotype individual vaccine-induced peptide-specific T-cell responses from cryopreserved PBMC. After the second MVA-CMDR boost, 73% (32 of 44) of vaccinees had higher frequencies of Gag-specific IFN-γ+ CD8+ T cells than after the first MVA boost, suggesting that optimal induction of Gag-specific CD8 T-cell responses probably needs a second MVA boost.
One limitation of this study is that cryopreservation negatively affected our ability to detect vaccine-induced T-cell responses. We did not observe this phenomenon during previous studies for natural HIV- or Mycobacterium tuberculosis-specific T-cell responses (22, 27). However, this phenomenon is not unknown (28).
In conclusion, our results show that after one MVA-CMDR boost, the sequence-mismatched TaMoVac 01 DNA prime/MVA boost vaccine regimen induced Gag-specific T-cell responses that were dominated by CD4+ T cells coexpressing IL-2 and TNF-α and targeted multiple conserved antigenic regions within Gagp24.
MATERIALS AND METHODS
Study design and samples.The TaMoVac 01 phase 2a trial was described in detail previously (16). Briefly, this randomized controlled trial was performed at Muhimbili University of Health and Allied Science (MUHAS) and the National Institute for Medical Research–Mbeya Medical Research Center (NIMR-MMRC) in Mbeya, Tanzania, with a total of 120 healthy, HIV-negative individuals, aged 18 to 40 years. Immunosuppressive medications were exclusion criteria. The TaMoVac 01 trial participants received two or five intradermal injections of DNA/placebo at weeks 0, 4, and 12 and were boosted with two MVA-CMDR/placebo intramuscular injections at weeks 30 and 46. The DNA/placebo was administered intradermally in the skin over both deltoids by using a Biojector 2000 needleless device (Bioject Medical Technologies, Inc., Tualatin, OR, USA), and the MVA injections were administered into the left deltoid muscle.
The DNA-SMI vaccine (Vecura, Huddinge, Stockholm Sweden) was composed of 7 plasmids encoding the HIV-1 genes Env (subtypes A, B, and C, respectively), Rev clade B, Gag (subtypes A and A/B), and reverse transcriptase subtype B (for details, see reference 29) and was administered at either 600 μg or 1,000 μg total. The recombinant modified vaccinia virus Ankara (MVA) expressing HIV-1 gp150 clade E as well as gag and pol clade A (MVA-CMDR), was manufactured by the Walter Reed Army Institute of Research (WRAIR) (30) and administered at 108 PFU.
The study was reviewed and approved by the National Ethics Committee, Institutional Review Board (IRB) at the NIMR-MMRC and the MUHAS IRB, in compliance with national guidelines and institutional policies (ClinicalTrials registration no. ATM2010050002122368), and informed consent was obtained in accordance with the Declaration of Helsinki.
In the present study, samples collected at 2 weeks after the first MVA-CMDR vaccination, when vaccine-induced T-cell responses peaked, from 42 vaccine recipients at the National Institute for Medical Research–Mbeya Medical Research Center and who did not become HIV infected during the trial were used for analyses.
HIV genetic sequence analyses.HIV-1 Gag sequences included in the phylogenetic analyses were from 91 HIV-positive subjects from the Mbeya region and have the GenBank accession numbers FJ853501 to FJ85359 (22). The subtypes and recombinant forms were determined using the jpHMM-HIV tool (http://jphmm.gobics.de/submission_hiv).
Peptide antigen and peptide pool design.DNA-SMI-Gagp37 and MVA-CMDR-Gagp55 peptide sets consisted of 15- to 18-mer peptides overlapping by 11 amino acids and had a purity of >80% (JPT Peptide Technologies, Berlin, Germany). Individual peptide variants of identical length for a given Gag region were used to allow for direct comparison of T-cell responses targeting MVA- or DNA-encoded peptide variants and to prevent artifacts linked to intrapeptide epitope location (24, 31). Peptide maxipools, including all peptides for the DNA-SMI-Gagp37 and MVA-CMDR-Gagp55 immunogens, were used to study phenotypic and functional characteristics of vaccine-induced Gag-specific T-cell responses. Linear peptide pools were used to test T-cell responses against distinct antigenic regions (Fig. 2), using nine pools for MVA-CMDR-Gagp55 and 7 pools for DNA-SMI-Gagp37.
ICS.An 8-color ICS assay was performed on fresh PBMC stimulated in the presence of brefeldin A (5 μg/ml; Sigma-Aldrich) for 6 h with either MVA-CMDR-Gagp55 or DNA-SMI-Gagp37 maxipools (1 μg/ml/peptide), nothing (negative control), or the control antigens (CMVpp65 peptide pool [0.5 μg/ml/peptide] and Staphylococcus enterococcus toxin B [1 μg/ml; Sigma-Aldrich]). Stimulated PBMC were then stained with α-CD3 allophycocyanin (APC)-Cy7 (BD Biosciences Europe, Erembodegem, Belgium), α-CD4 peridinin chlorophyll protein (PerCp)-Cy5.5 (eBioscience, San Diego, CA), α-CD8 V500 (BD Bioscience), α-TNF-α phycoerythrin (PE)-Cy7 (BD), α-IFN-γ V450 (BD), α-IL-2 APC (BD), α-MIP-1β PE (BD), or α-CD107 fluorescein isothiocyanate (eBioscience). Acquisition of samples was performed using a FACSCanto II flow cytometer with acquisition-defined compensation using BD CompBeads (BD). Flow cytometry results were analyzed using FlowJo software, version 9.6.4 (Tree Star, Ashland, OR), and SPICE, version 5.35, downloaded from http://exon.niaid.nih.gov (32). A minimum of 50,000 CD3+ lymphocytes were required for a sample to be included in the analysis. Background reactivity, defined by using unstimulated negative controls, was subtracted for analyses of antigen-specific T-cell responses. IFN-γ+ T-cell frequencies of >0.025% were considered a positive response.
IFN-γ ELISPOT assay.The IFN-γ ELISPOT plus kit (Mabtech, Nacka, Sweden) was used according to the instructions of the manufacturer. Fresh peripheral blood mononuclear cells (PBMC) were stimulated with linear peptide pools matching DNA-SMI-Gagp37 and MVA-CMDR-Gagp55 (described below; JPT, Berlin, Germany) (Fig. 2). Frequencies of antigen-specific SFC were measured with an automated ELISPOT assay reader (ImmunoSpot CTL, Bonn, Germany). Responses were considered positive when the number of SFC was at least four times the medium control and >55 SFC/106 PBMC (33). Mapping of individual peptide responses was performed based on the peptide pool matrix ELISPOT assay results and testing cryopreserved PBMC. Responses of stimulated PBMC with an SFC count higher than 2-fold the unstimulated medium control and ≥50 SCF/106 PBMC cells were considered positive. Samples with a medium control of >60 SFC/PBMC were excluded from analyses.
Statistical analyses.Statistical analyses were performed using Prism version 6.0 (GraphPad, Inc.). Comparisons of two groups were performed with the Mann-Whitney test. Magnitudes of CD4 and CD8 T-cell responses were performed with the Wilcoxon signed-rank test. Number of responders with IFN-γ+ CD4 and CD8 T-cell responses were compared using Fisher's exact test. The linear relationship between the number of mismatched amino acid positions within a given antigenic region and the corresponding frequency of recognition and response magnitude was calculated using linear regression analyses. Tests used for statistical analysis are mentioned in the figure legends.
ACKNOWLEDGMENTS
We gratefully acknowledge funding from the European and Developing Countries Clinical Trials Partnership (EDCTP), grant CT.2006.33111.007, from the German Ministry of Education and Research (BMBF, grant 01KA0802), and from the German Center for Infection Research (DZIF). This work was supported by the Medical Research Council (MRC_UU_12023/23).
The funders had no role in study design, data collection and interpretation, or the decision to submit the work for publication.
We have no conflicts of interest to declare.
FOOTNOTES
- Received 18 May 2017.
- Accepted 14 June 2017.
- Accepted manuscript posted online 12 July 2017.
- Copyright © 2017 American Society for Microbiology.
