Immunogenicity of Seven New Recombinant Yellow Fever Viruses 17D Expressing Fragments of SIVmac239 Gag, Nef, and Vif in Indian Rhesus Macaques

<div><p>An effective vaccine remains the best solution to stop the spread of human immunodeficiency virus (HIV). Cellular immune responses have been repeatedly associated with control of viral replication and thus may be an important element of the immune response that must be evoked by an efficacious vaccine. Recombinant viral vectors can induce potent T-cell responses. Although several viral vectors have been developed to deliver HIV genes, only a few have been advanced for clinical trials. The live-attenuated yellow fever vaccine virus 17D (YF17D) has many properties that make it an attractive vector for AIDS vaccine regimens. YF17D is well tolerated in humans and vaccination induces robust T-cell responses that persist for years. Additionally, methods to manipulate the YF17D genome have been established, enabling the generation of recombinant (r)YF17D vectors carrying genes from unrelated pathogens. Here, we report the generation of seven new rYF17D viruses expressing fragments of simian immunodeficiency virus (SIV)mac239 Gag, Nef, and Vif. Studies in Indian rhesus macaques demonstrated that these live-attenuated vectors replicated <em>in vivo,</em> but only elicited low levels of SIV-specific cellular responses. Boosting with recombinant Adenovirus type-5 (rAd5) vectors resulted in robust expansion of SIV-specific CD8⁺ T-cell responses, particularly those targeting Vif. Priming with rYF17D also increased the frequency of CD4⁺ cellular responses in rYF17D/rAd5-immunized macaques compared to animals that received rAd5 only. The effect of the rYF17D prime on the breadth of SIV-specific T-cell responses was limited and we also found evidence that some rYF17D vectors were more effective than others at priming SIV-specific T-cell responses. Together, our data suggest that YF17D – a clinically relevant vaccine vector – can be used to prime AIDS virus-specific T-cell responses in heterologous prime boost regimens. However, it will be important to optimize rYF17D-based vaccine regimens to ensure maximum delivery of all immunogens in a multivalent vaccine.</p> </div

Functional profile of CD8⁺ cellular responses in rYF17D/rAd5 and rAd5 vaccinees.

<p>We carried out multi-parameter ICS at week 3 after the rAd5 immunization to determine the ability of SIV-specific CD8⁺ T-cells to degranulate (CD107a) and secrete IFN-γ, TNF-α, MIP-1β, and IL-2. The antigen stimuli in this assay consisted of peptide mixtures spanning (i) amino acids 1–291 of Gag, (ii) amino acids 281–510 of Gag, (iii) the Vif ORF, and (iv) the Nef ORF. Bar graphs indicate the mean total frequency of CD8⁺ lymphocytes specific to Gag, Nef, and Vif capable of producing each combination of functions tested. Pie graphs for each animal indicate the percentage of their CD8⁺ lymphocytes that are specific to Gag, Nef, and Vif and that were positive for one (yellow), two (green), three (orange), four (red), and five (black) immune parameters. A) Functional profile of CD8⁺ cellular responses in rYF17D/rAd5 vaccinees. B) Functional profile of CD8⁺ cellular responses in rAd5 vaccinees. Error bars represent the standard error of the mean.</p

Magnitude of SIV-specific CD4⁺ and CD8⁺ cellular responses in rYF17D/rAd5 and rAd5 vaccinees.

<p>At one week after the rAd5 vaccination, we obtained PBMC from all animals and performed ICS to determine the frequency of IFN-γ-producing CD8⁺ (A and B) and CD4⁺ (C and D) lymphocytes specific to Gag (black bars), Nef (gray bars), Vif (plaid bars), and the combination of Tat, Rev, Vpr, and Vpx (white bars). The antigen stimuli in this assay consisted of peptide mixtures spanning (i) amino acids 1–291 of Gag, (ii) amino acids 281–510 of Gag, (iii) the Vif ORF, (iv) the Nef ORF, (v) the Tat ORF, (vi) the Rev ORF, and (vii) the Vpr and Vpx ORFs. Reactivity to the Gag protein is reported as the sum of tests (i) and (ii); reactivity to Tat, Rev, Vpr, and Vpx is reported as the sum of tests (v), (vi), and (vii). A and C show the results for the rYF17D/rAd5 group while B and D show results for the rAd5 group.</p

Magnitude of SIV-specific T-cell responses in animals vaccinated with single rYF17D/SIV constructs.

<p>We carried out IFN-γ ELISPOT at days 14 (white bars) and 17 (black bars) after the rYF17D vaccination using peptide pools (ten 15mers overlapping by 11 amino acids in each pool) spanning the regions of SIVmac239 Gag, Vif, and Nef encoded in each rYF17D vector. We adjusted these pools for each macaque depending on the region and SIV protein expressed by the rYF17D viruses. Bar graphs indicate the magnitude of IFN-γ-producing cells in PBMC (SFC/10⁶ PBMC) in macaques immunized with rYF17D/Gag constructs (A), rYF17D/Vif constructs (B), and the rYF17D/Nef construct (C). Responses measured at days 14 and 17 following vaccination with the rYF17D/SIV vectors are shown by white and black bars, respectively. Of note, the vaccine candidates rYF17D/Gag(44–84), rYF17D/Vif(1–110), and rYF17D/Nef(45–210) encode three Mamu-A*02-restricted, CD8⁺ T-cell epitopes: Gag_71–79GY9, Vif_97–104WY8, and Nef_159–167YY9, respectively <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0054434#pone.0054434-Loffredo2" target="_blank">[50]</a>, <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0054434#pone.0054434-Vogel1" target="_blank">[51]</a>. Since the recipients of these vectors were <i>Mamu-A*02⁺</i> (r04147, r05089, and r05070; <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0054434#pone-0054434-t001" target="_blank">Table 1</a>), we used minimal optimal peptides in their IFN-γ ELISPOT assays to determine the frequency of CD8⁺ T-cells recognizing the Gag_71–79GY9, Vif_97–104WY8, and Nef_159–167YY9 epitopes. We also assessed vector-specific cellular responses by using synthetic peptides corresponding to four Mamu-A*01-restricted CD8⁺ T-cell epitopes in the backbone of YF17D identified in a previous study <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0054434#pone.0054434-Bonaldo3" target="_blank">[37]</a>. The amino acid sequence of these peptides and their corresponding position in the YF17D polyprotein are as follows: LTPVTMAEV (LV9_1285–1293), VSPGNGWMI (VI9_3250–3258), MSPKGISRM (MM9_2179–2187), and TTPFGQQRVF (TF10_2853–2862). We measured responses to these four epitopes in the <i>Mamu-A*01⁺</i> macaques r04170, r04136, r05079, and r04109. An asterisk (*) on top of a bar indicates a positive response detected in CD8-depleted PBMC, which likely represents SIV-specific CD4⁺ T-cells. The number of IFN-γ-producing cells in positive control wells stimulated with Concanavalin A at days 14 and 17 were 13,321 and 6,194 SFC/10⁶ PBMC, respectively.</p

Animal details and vaccination doses.

<p>N.A., not applicable.</p

Breadth of vaccine-induced, SIV-specific T-cell responses in rYF17D/rAd5 and rAd5 vaccinees.

<p>We determined the breadth of T-cell responses in each animal by adding the number of positive IFN-γ ELISPOT assay responses to peptide pools (15mers overlapping by 11 amino acids) spanning the regions of Gag, Nef, and Vif covered in the SIVmac239 inserts. Panels A and B show the median breadth of T-cell responses to Gag, Nef, Vif, and all three proteins combined in the rYF17D/rAd5 (A) and rAd5 (B) groups. C) Comparison of the total number of peptide pools recognized in rYF17D/rAd5- and rAd5-vaccinated animals. Lines represent the median total number of responses in each group. We used the Mann-Whitney test to determine the p value.</p

Relative sizes of SIV inserts and genome schematic of rYF17D/SIV vectors.

<p>A) Relative size and amino acid position of SIVmac239 minigenes. We designed codon-optimized sequences spanning regions of the Gag, Vif, and Nef proteins. The Gag minigenes covered amino acids 44–84 and 76–123 of Matrix (MA), 142–186 of Capsid (CA), and 250–415 of Capsid, p2, and Nucleocapsid (NC). The Vif ORF was split into two minigenes encoding amino acids 1–110 and 102–214. The Nef sequence spanned the central region of this protein (45–210). B) Genome schematic of recombinant YF17D vaccine vectors. We inserted the SIVmac239 minigenes in the junction between the YF17D E and NS1 genes using a previously described methodology <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0054434#pone.0054434-Bonaldo2" target="_blank">[36]</a>. C) Electrophoretic analysis of RT-PCR amplicons from viral RNA extracted from the seven rYF17D/SIV stocks. The YF17D parental vaccine served as a negative control. We performed these amplifications using YF17D-specific primers flanking the E-NS1 genomic region encompassing the SIVmac239 inserts.</p

Kinetics of vaccine-induced, SIV-specific T-cell responses in rYF17D/rAd5 and rAd5 vaccinees.

<p>We used IFN-γ ELISPOT to determine the magnitude of vaccine-induced T-cell responses at weeks 1, 2, 3, and 6 following the rAd5 immunization. The median frequencies of T-cell responses to Vif (A), Nef (B), Gag (C), and the combination of Tat, Rev, Vpr, and Vpx (D) are shown for rYF17D/rAd5 (black circles) and rAd5 (white circles) vaccinees. We determined the magnitude of responses to each protein by adding SFC/10⁶ PBMC values obtained in test wells containing peptide pools spanning the SIV inserts described in <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0054434#pone-0054434-g001" target="_blank">Figure 1A</a>. Error bars represent the interquartile range. Statistical comparisons were made using the Mann-Whitney test. The # symbol in the Vif graph indicates a p-value of 0.06 while asterisks (*) denote p<0.05. The average number of IFN-γ-producing cells in positive control wells stimulated with Concanavalin A at these time points ranged between 4,394 and 14,701 SFC/10⁶ PBMC.</p

Magnitude of SIV-specific T-cell responses in animals vaccinated with all seven rYF17D/SIV constructs.

<p>We used the same approach described in the legend of <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0054434#pone-0054434-g002" target="_blank">Figure 2</a> to measure SIV-specific T-cell responses in animals that were vaccinated with all seven rYF17D/SIV viruses. The only difference was that the IFN-γ ELISPOT assays for these animals contained all peptide pools spanning the regions encoded in the Gag, Vif, and Nef inserts. We performed these analyses at days 14 (white bars) and 17 (black bars) after vaccination with rYF17D/SIV. Vaccinees r05028 and rh2138 received 10⁴ PFU of each vaccine vector (A) while r04137 and r98010 received 10⁵ PFU of each construct (B). Similar to the description in the legend of <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0054434#pone-0054434-g002" target="_blank">Figure 2</a>, we measured the frequency of SIV-specific CD8⁺ T-cells in <i>Mamu-A*02⁺</i> macaques by using minimal optimal peptides corresponding to the three Mamu-A*02-restricted epitopes Gag_71–79GY9, Vif_97–104WY8, and Nef_159–167YY9. We also determined the magnitude of vector-specific CD8⁺ T-cells in the <i>Mamu-A*01⁺</i> macaques r05028 and r04137 by using synthetic peptides corresponding to the YF17D epitopes LV9_1285–1293, VI9_3250–3258, MM9_2179–2187, and TF10_2853–2862. An asterisk (*) on top of a bar indicates a positive response detected in CD8-depleted PBMC, which likely represents SIV-specific CD4⁺ T-cells. The average number of IFN-γ-producing cells in positive control wells stimulated with Concanavalin A at days 14 and 17 were 10,980 and 9,269 SFC/10⁶ PBMC, respectively.</p