Mosaic vaccines elicit CD8 + T lymphocyte responses that confer enhanced immune coverage of diverse HIV strains in monkeys

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1 Mosaic vaccines elicit CD + T lymphocyte responses that fer enhanced immune coverage of diverse HIV strains in monkeys 21 Nature America, Inc. All rights reserved. Sampa Santra 1, Hua-Xin Liao 2, Ruijin Zhang 2, Mark Muldoon 3, Sydeaka Watson,, Will Fischer, James Theiler, James Szinger, Harikrishnan Balachandran 1, Adam Buzby 1, David Quinn 1, Robert J Parks 2, Chun-Yen Tsao 2, Angela Carville 6, Keith G Mansfield 6, George N Pavlakis 7, Barbara K Felber 7, Barton F Haynes 2, Bette T Korber, & Norman L Letvin 1 An effective HIV vaccine must elicit immune responses that recognize genetically diverse viruses 1,2. It must generate CD + T lymphocytes that trol HIV replication and CD + T lymphocytes that provide help for the generation and maintenance of both cellular and humoral immune responses against the virus 3. Creating immunogens that can elicit cellular immune responses against the genetically varied circulating isolates of HIV presents a key challenge for creating an HIV vaccine 6,7. Polyvalent mosaic immunogens derived by in silico recombination of natural strains of HIV are designed to induce cellular immune responses that recognize genetically diverse circulating virus isolates. Here we immunized rhesus monkeys by plasmid DNA prime and recombinant vaccinia virus boost with vaccine structs expressing either sensus or polyvalent mosaic proteins. As compared to sensus immunogens, the mosaic immunogens elicited CD + T lymphocyte responses to more epitopes of each viral protein than did the sensus immunogens and to more variant sequences of CD + T lymphocyte epitopes. This increased breadth and depth of epitope recognition may tribute both to protection against infection by genetically diverse viruses and to the trol of variant viruses that emerge as they mutate away from recognition by cytotoxic T lymphocytes. Mosaic immunogen sequences are designed with an algorithm that simulates evolution by recombination, using coverage of potential T lymphocyte epitopes as the selection criterion. Nine-amino-acid fragments are sidered potential epitopes, as nine amino acids is the most common length of major histocompatibility complex (MHC) class I presented peptides and the most common span between anchor residues of MHC class II presented peptides 9. These sequences form complete proteins and are created with recombination breakpoints that are found in nature. We initiated this study to explore the breadth of CD + and CD + T lymphocyte responses generated through vaccination with polyvalent mosaic and sensus immunogens. We selected two HIV genes for use in the immunogens employed in this study : HIV-1 gag and nef. We separated 3 rhesus monkeys into three cohorts, two experimental groups sisting of monkeys each and a trol group sisting of six monkeys. One experimental group received mosaic immunogens, the other experimental group received sensus immunogens, and the trol group received empty vectors. We chose a DNA prime recombinant vaccinia virus boost regimen for this study, as these immunogens elicit both CD + and CD + T lymphocyte responses 1. We determined the breadth of the vaccine-elicited cellular immune responses by assessing peripheral blood T lymphocyte recognition of ten natural and sequences with a peptide-stimulated interferon-γ (IFN-γ) enzyme-linked immunospot (ELISPOT) assay and matrix epitope mapping. The and sets of ten indicator proteins included two clade A, two clade B, four clade C and two clade G sequences (a clade of HIV-1 sequences is a group of genetically related, usually geographically discrete, virus isolates). We used the same set of sequences that we had previously used to evaluate vaccineelicited responses to a sensus Env immunogen 13. We selected these isolate sequences to represent recently sampled viruses, diverse geographic origins and genetically distinct subregions of each clade. This strategy allowed us to assess vaccine-induced responses to sequences representative of the diverse HIV-1 strains to which human vaccinees will be exposed. Many nonamers in an HIV-1 strain are rare in the overall population of viruses (% of nonamers and 67% of nonamers are present in <% of wild-type sequences, on the basis of the Los Alamos database collection of M group sequences). As rare epitope variants are such a large component of the potential response, it is useful to know how often a vaccine can induce 1 Beth Israel Deaess Medical Center, Harvard Medical School, Boston, Massachusetts, USA. 2 Duke University Medical Center, Durham, North Carolina, USA. 3 University of Manchester School of Mathematics, Manchester, UK. Los Alamos National Laboratory, Los Alamos, New Mexico, USA. Departmental of Statistical Sciences, Baylor University, Waco, Texas, USA. 6 New England Primate Research Center, Southborough, Massachusetts, USA. 7 National Cancer Institute, US National Institutes of Health, Frederick, Maryland, USA. Santa Fe Institute, Santa Fe, New Mexico, USA. Correspondence should be addressed to N.L.L. (nletvin@bidmc.harvard.edu). Received 2 September 29; accepted 27 January 21; published online 21 February 21; doi:1.13/nm VOLUME 16 NUMBER 3 MARCH 21 nature medicine

2 21 Nature America, Inc. All rights reserved. Figure 1 Number of epitope-specific responses by CD + and CD + T lymphocytes of vaccinated monkeys. (a) CD + and CD + T lymphocyte responses from seven monkeys per group were assessed for their recognition of specific epitopes of each of the ten indicator and proteins in IFN-γ ELISPOT assays. Unfractionated and CD + lymphocyte-depleted PBLs were assessed for their recognition of specific epitope peptides. (b) The breadth of CD + and CD + T lymphocyte responses by individual monkeys was determined as follows: if a -mer peptide from one of the ten sets of indicator proteins was recognized by a T lymphocyte population, it was scored as one positive; if multiple variant forms of the same peptide from more than one set of indicator proteins were recognized by a T lymphocyte population, they were also scored as one positive response. (c) The depth of CD + and CD + T lymphocyte responses by individual monkeys, as determined by counting the responses made to all variant peptides from each epitopic region of either the or the protein. responses to rare nonamers in representative strains. This peptide design strategy allowed us to determine the precise number of vaccine-elicited responses per virus strain that were induced in each monkey and to characterize the cross-reactive potential of each epitope-specific response. The magnitudes of the vaccine-induced -specific and -specific antibody and ELISPOT responses between the groups of monkeys were comparable (Supplementary Fig. 1a c); therefore, any detected differences in the cross-reactivity of the cellular responses between these groups of vaccinated monkeys were not simply a manifestation of a more robust general immune response. The median number of epitope-specific total T lymphocyte responses generated by each monkey against and from a single virus strain after vaccination with sensus immunogens was. (interquartile range ); the median number per strain after vaccination with mosaic immunogens was 7. (interquartile range ). There was no statistically significant difference between these numbers of responses in the two experimental groups of monkeys (Wilcoxon rank-sum test). To assess whether one of the vaccine strategies induced cellular immune responses that recognized a greater diversity of epitope variants, we determined the average number of variants recognized per epitope-specific response by dividing the total number of variant peptides recognized by the number of epitope-specific responses in each vaccinated monkey. The mosaic-vaccinated monkeys had higher ratios than the sensus-vaccinated monkeys (P =., Wilcoxon rank-sum test; Supplementary Table 1). The vectors used in the present immunizations generated higher levels of CD + than of CD + T lymphocyte immune responses, and any benefit for CD + T lymphocyte responses derived from using mosaic immunogens might be obscured in the overall response by the CD + T lymphocyte responses 1. As CD + T lymphocytes recognize peptide antigens associated with MHC class II molecules, and peptide MHC class II binding is more promiscuous than peptide MHC class I a Number of epitopes per strain b Number of epitopes per monkey c Number of variant peptides per monkey Mosaic, CD Mosaic, CD 7 Consensus, CD 7 Mosaic, CD 7 Consensus, CD Mosaic, CD Consensus, CD Consensus, CD Mosaic, CD Mosaic, CD Consensus, CD Consensus, CD binding, benefits associated with mosaic immunogens might be more apparent in CD + than CD + T lymphocyte responses,16. To determine the relative impact of mosaic and sensus immunogens on CD + and CD + T lymphocyte responses separately, we evaluated the breadth of responses to HIV and that were generated by mosaic and sensus gene-based immunizations using unfractionated and CD + T lymphocyte depleted peripheral blood lymphocyte (PBL) populations. Sufficient lymphocytes were available from seven monkeys in each of the vaccinated groups to carry out this evaluation. We determined the total number of epitope-specific responses to all tested strains of and by PBLs of each monkey and compared the groups with the Wilcoxon rank-sum test (Fig. 1a). The median number of epitope-specific CD + T lymphocyte responses per strain induced by vaccination of the monkeys with the mosaic immunogens was. (interquartile range 2. 7.), and it was 2. (interquartile range 1.2.) in monkeys vaccinated with the sensus immunogens. The median number of epitope-specific CD + T lymphocyte responses per strain induced by vaccination of the monkeys with the mosaic immunogens was 2. (interquartile range 1. 3.), and it was 1. with the sensus immunogens (interquartile range. 1.) (Supplementary Fig. 2 tains a full breakdown of responses by protein and by CD + and CD + T lymphocytes) A1, A2 B2, B2 C1, C2, C3, C G1, G2 nature medicine VOLUME 16 NUMBER 3 MARCH 21 32

3 21 Nature America, Inc. All rights reserved. a b c d sensus _A2 _A1_G2 _B1 _B2 _C1 _C2 _C3 _C _G1 sensus _A1_A2_B2_C1_C3_C _B1_C2_G1_G2 _A1_A2_B2_C1_C3 _B1_C2_G1_G2 _C sensus Mosaic 1 Mosaic 2 Mosaic 3 Mosaic _A2 _A1_B2 _B1 _C1 _C2 _C3 _C _G1 _G2 sensus Mosaic 1 Mosaic 2 Mosaic 3 Mosaic _A1 _B1_B2 _C1 _C3 _A2 _C2_C _G1 _G2 mos1 C1 M1 There were more total T lymphocyte responses (CD + plus CD + ) per strain induced by mosaic immunogens than by sensus immunogens by a factor of two (P =.23, Poisson regression), but vaccine-induced responses did not differ significantly between the two groups. We then compared the number of epitope-specific CD + and CD + T lymphocyte responses per monkey, counting overlapping peptides or sequence variants as one event (Fig. 1b). There was no significant difference in the number of total (-specific plus -specific) (Wilcoxon rank-sum statistics; P =.2), -specific (P =.1) or -specific (P =.) CD + T lymphocyte responses elicited by sensus or mosaic immunogens (Fig. 1b). However, mosaic immunogens induced a significantly larger number of total (P =.1) and -specific (P =.6) and a trend toward more -specific (P =.1) CD + T lymphocyte responses. We also assessed whether mosaic-immunized monkeys developed CD + and CD + T lymphocyte responses with greater depth than sensus-immunized monkeys by quantifying the number of responses to all of the variant forms of a given epitope (Fig. 1c). There were more -specific (P =.) but not -specific (P =.) CD + T lymphocyte responses to variant peptides per response in the mosaic-immunized group; there was a median number of 1 (range 1 3) epitope specific CD + T lymphocyte response in the sensus-immunized monkeys and a median number of 3 (range 2 ) of such responses in the mosaic-immunized monkeys (Fig. 1c). For CD + T lymphocyte responses, there were more total (-specific plus -specific) responses (P =.), -specific responses (P =.1) and a trend toward more -specific responses (P =.16) in the mosaic-immunized monkeys than in the sensus-immunized monkeys (Fig. 1c). These findings are comparable to those reported M2 Susceptible variant Nonsusceptible variant C C16 Figure 2 Examples of the depth of vaccine-elicited CD + T lymphocyte responses. Consensus and mosaic vaccine-induced CD + T lymphocyte responses were assessed for recognition of variant forms of the same region of a viral protein. For each example, the variant HIV-1 sequences are shown aligned to the M group sensus of that sequence. Amino acid identity to the sensus sequence is indicated by a dash. For some sequences, a blank space is inserted to maintain the alignment. The peptides recognized by PBLs of a vaccinated monkey are shown in black at the top and are preceded by the number of the responding monkey. The variant peptides in the same region that are not recognized are shown in red. Every unique peptide sequence recognized by PBLs is shown in a different shade of green, and white boxes represent peptides that are not recognized. A large box represents an exact match of a number of sequences. (a) A highly restricted CD + T lymphocyte response. CD + T lymphocytes from monkey recognize only the peptide sequence that matches the vaccine sequence. (b) A cross-reactive CD + T lymphocyte response. Three different variants of peptide and 16 sequences exist in ten indicator proteins, and CD + T lymphocytes from monkey recognize all three variants. (c) A highly restricted CD + T lymphocyte response. CD + T lymphocytes of monkey recognize only the variant peptide that matches one of the four mosaic sequences used in the mosaic immunogen cocktail. (d) A cross-reactive CD + T lymphocyte response. CD + T lymphocytes from monkey recognize four different variant forms of the peptide, all of which differ in sequence from the vaccine immunogens. by Barouch et al. 17 in this issue (a median of 1 (range 2) in the sensus-immunized monkeys and a median of 3 (range 2 ) in the mosaic-immunized monkeys). Together, these data suggest that the benefit of the mosaic immunogens was more apparent for CD + T lymphocyte than for CD + T lymphocyte responses. Because we evaluated the reactivity of the PBLs of each vaccinated monkey to peptides spanning the entire and proteins of ten genetically disparate HIV-1 isolates, we were able to define the immune recognition of actual proteins from distinct circulating virus strains in the immunized monkeys. Examples of typical responses and our approach to illustrating them are shown in Figure 2; the complete alignment of all peptides to which at least a single CD + T lymphocyte response was made is shown in Supplementary Figure 3. Some vaccine-induced CD + T lymphocyte responses were highly strain specific, capable of recognizing peptide variants from only one or two of the ten test strains (Figs. 2a,c and 3). We observed broadly cross-reactive responses to individual epitopes, but they were very rare, with all ten peptide variants recognized only six times in all vaccinated monkeys (Figs. 2b and 3). There were some responses to two overlapping peptides, probably representing the recognition of a single epitope (Fig. 2b). CD + T lymphocyte responses were usually highly specific. Extending the breadth of responses to more than two variant peptides was rare in both groups of vaccinated monkeys. This type of potentially valuable response occurred only six times among the sensus vaccine induced CD + T lymphocyte responses and 17 times among responses in mosaic vaccine recipients (binomial test P =.3; Figs. 2d and 3). Modeling the depth of the peptide-specific responses as a function of vaccine and T lymphocyte type, we found that mosaic vaccine elicited CD + T lymphocyte responses were more likely than sensus vaccine elicited CD + T lymphocyte responses to recognize variant peptides by a factor of 1.6 (P =.6, binomial regression; Fig. 3 and Supplementary Fig. ). Extending the breadth of a response to more than two variant peptides occurred 2 times among CD + T lymphocyte responses in the mosaic immunized monkeys and only 23 times in the sensus-vaccinated monkeys (binomial test P =.; Fig. 3 and Supplementary Figs. and 6). CD + T lymphocyte 326 VOLUME 16 NUMBER 3 MARCH 21 nature medicine

4 Figure 3 Breadth and depth of - and -specific CD + T lymphocyte responses in seven monkeys from each vaccine group. All of the and peptides recognized by PBLs of individual monkeys were aligned to the HIV-1 M group sensus sequences as described in the legend of Figure 2. Lymphocyte recognition of any peptide sequence is shown in rectangles of shades of green, orange or yellow, and nonrecognition of a peptide is shown in unshaded rectangles. M9 M M1 M1 M6 M19 M16 M7 M2 Mosaic M17 M M3 M32 M13 M36 M33 M1 M3 M3 M M M M1 M2 11 C1 C C3 C2 C1 C11 Consensus C13 C C1 C1 C6 C19 C7 21 Nature America, Inc. All rights reserved. responses usually had greater depth (encompass a larger fraction of variants) than CD + T lymphocyte responses by a factor of 1. (P =.); this was true for both vaccine prototypes and may explain why the benefit of mosaic immunogens was more pronounced for CD + T lymphocytes. Almost all of the indicator strains of and proteins were recognized by one or two epitope-specific CD + T lymphocyte responses in the PBLs of both groups of vaccinated monkeys (Fig. a). However, we detected more CD + T lymphocyte responses that recognized three epitopes in mosaic-vaccinated monkeys than in sensus-vaccinated monkeys. Almost all of the indicator strains of peptides were recognized by at least one epitope-specific CD + T lymphocyte response in the PBLs of the mosaic vaccine recipients, whereas a substantial number of viral strains were not recognized by epitope-specific CD + T lymphocyte peptide responses in the sensus-immunized monkeys (P =.7, Wilcoxon rank sum test; Fig. b). The mosaic immunogens also elicited a significantly larger number of CD + T lymphocyte responses that recognized a minimum of two (P =.22) and three epitopes (P =.1) of these natural virus strains as compared to the sensus immunogen. In the recent STEP human vaccine trial, an adenovirus-based HIV-1 -Pol- vaccine induced a median of only two epitope-specific responses to the vaccine in each subject (N. Frahm, personal communication), only % of the subjects mounted a response to M11 M3 M1 M M22 M2 M39 M2 M21 M23 M2 M M3 M7 M3 M26 M1 M M M M27 M2 M9 M1 M2 M M11 M29 M6 M M3 M13 M31 M1 M37 M Nonsusceptible variants Susceptible variants, and only 32% of vaccinees generated both a CD + and a CD + T lymphocyte response 1. It is possible that this vaccine failed to fer protection because most of the vaccine-elicited responses were not able to recognize the circulating strains of HIV-1. Our study demonstrates that immunization of nonhuman primates with mosaic immunogens induces CD + T lymphocytes with greater cross-reactivity than the sensus immunogen. The mosaic immunogens elicited CD + T lymphocyte responses to more epitopes of each viral protein than the sensus immunogens and to more variant sequences of the CD + T lymphocyte epitopes. This increased breadth and depth of epitope recognition could tribute to protection against infection by genetically diverse viruses and, in some instances, may block the emergence of common variant viruses. C7 C C9 C C C16 C1 C6 C17 C2 C C3 C C a CD + epitopes Mosaic Consensus Mosaic Consensus min 1 min 2 min 3 min 1 min 2 min 3 min 1 min 2 min 3 min 1 min 2 min 3 11 b CD + epitopes 11 G2 G1 C C3 C2 C1 B2 B1 A2 A1 Figure Cross-clade epitope recognition by CD + and CD + T lymphocytes of seven monkeys from each vaccine group. (a,b) The stacks of ten rectangles represent the ten sets of indicator peptides with A1 and A2 shown in turquoise; B1 and B2 in red; C1, C2, C3 and C in purple; and G1 and G2 in dark blue. For each monkey, in the left-hand column (min 1), if at least one peptide from an indicator peptide set was recognized by either CD + (a) or CD + (b) T lymphocytes, the rectangle is shaded with its representative color. If no PBL responses to a particular set of indicator peptides were detected, the rectangle is shown unshaded. Recognition of one, two or three epitopes per strain of indicator proteins by CD + and CD + T lymphocytes of each of the seven monkeys in both vaccine groups is shown as min 1, min 2 and min 3, respectively. nature medicine VOLUME 16 NUMBER 3 MARCH

5 21 Nature America, Inc. All rights reserved. Methods Methods and any associated references are available in the online version of the paper at Note: Supplementary information is available on the Nature Medicine website. Acknowledgments We thank G. Tomaras for generous advice and assistance with reagents and antibody data. We also thank C. Lord for assistance with reagents. This work was supported by US National Institutes of Health grant AI-671 (Center for HIV AIDS Vaccine Immunology), HIV Research and Design (HIVRAD) P1 AI-6173, National Institute of Allergy and Infectious Diseases tract N1 AI-1 and a Los Alamos National Laboratory directed research grant to B.T.K., W.F. and S.W. AUTHOR CONTRIBUTIONS B.T.K., W.F., G.N.P. and B.K.F. designed the vaccine gene inserts. H.-X.L., R.Z. and B.F.H. generated the recombinant vaccinia virus structs. B.T.K., B.F.H., S.S. and N.L.L. designed the study. A.C. and K.G.M. performed all of the immunizations. H.B., A.B., D.Q. and S.S. designed and ducted the cellular immunologic assays. R.J.P., C.-Y.T. and B.F.H. designed and ducted the antibody assays. M.M., S.W., W.F., J.T., J.S. and B.T.K. performed the data analysis. COMPETING INTERESTS STATEMENT The authors declare no competing financial interests. Published online at Reprints and permissions information is available online at reprintsandpermissions/. 1. Korber, B.T., Letvin, N.L. & Haynes, B.F. T-cell vaccine strategies for human immunodeficiency virus, the virus with a thousand faces. J. Virol. 3, 3 31 (29). 2. Barouch, D.H. Challenges in the development of an HIV-1 vaccine. Nature, (2). 3. Letvin, N.L. Progress and obstacles in the development of an AIDS vaccine. Nat. Rev. Immunol. 6, 9 (26).. Heeney, J.L. Requirement of diverse T-helper responses elicited by HIV vaccines: induction of highly targeted humoral and CTL responses. Expert Rev. Vaccines 3, S3 S6 (2).. McMichael, A.J. HIV vaccines. Annu. Rev. Immunol. 2, (26). 6. Fischer, W., Liao, H.X., Haynes, B.F., Letvin, N.L. & Korber, B. Coping with viral diversity in HIV vaccine design: a response to Nickle et al. PLoS Comput. Biol., e (2). 7. Gaschen, B. et al. Diversity siderations in HIV-1 vaccine selection. Science 296, (22).. Fischer, W. et al. Polyvalent vaccines for optimal coverage of potential T-cell epitopes in global HIV-1 variants. Nat. Med. 13, 1 16 (27). 9. Rammensee, H., Bachmann, J., Emmerich, N.P., Bachor, O.A. & Stevanovic, S. SYFPEITHI: database for MHC ligands and peptide motifs. Immunogenetics, (1999). 1. Santra, S. et al. Recombinant poxvirus boosting of DNA-primed rhesus monkeys augments peak but not memory T lymphocyte responses. Proc. Natl. Acad. Sci. USA 11, (2). 11. Precopio, M.L. et al. Immunization with vaccinia virus induces polyfunctional and phenotypically distinctive CD + T cell responses. J. Exp. Med. 2, (27).. Harari, A. et al. An HIV-1 clade C DNA prime, NYVAC boost vaccine regimen induces reliable, polyfunctional and long-lasting T cell responses. J. Exp. Med. 2, (2). 13. Santra, S. et al. A centralized gene-based HIV-1 vaccine elicits broad cross-clade cellular immune responses in rhesus monkeys. Proc. Natl. Acad. Sci. USA, (2). 1. Kong, W.P. et al. Expanded breadth of the T-cell response to mosaic human immunodeficiency virus type 1 envelope DNA vaccination. J. Virol. 3, (29).. Moss, C.X., Tree, T.I. & Watts, C. Restruction of a pathway of antigen processing and class II MHC peptide capture. EMBO J. 26, (27). 16. Sette, A., Adorini, L., Colon, S.M., Buus, S. & Grey, H.M. Capacity of intact proteins to bind to MHC class II molecules. J. Immunol. 13, 67 (199). 17. Barouch, D.H. et al. Mosaic HIV-1 vaccines expand the breadth and depth of cellular immune responses in rhesus monkeys. Nat. Med. advance online publication, doi:1.13/nm.29 (21 February 21). 1. McElrath, M.J. et al. HIV-1 vaccine-induced immunity in the test-of-cept Step Study: a case-cohort analysis. Lancet 372, 19 1 (2). 32 VOLUME 16 NUMBER 3 MARCH 21 nature medicine

6 21 Nature America, Inc. All rights reserved. ONLINE METHODS Experimental groups and vaccination schedule. We housed the monkeys at the New England Regional Primate Research Center. We maintained the monkeys in accordance with standards set forth by American Association for Accreditation of Laboratory Animal Care and the study protocols were approved by the Harvard University Medical School Institutional Animal Care and Use Committee. We distributed 3 Mamu-A*1, Mamu-B*1 and Mamu-B*17 negative rhesus monkeys into three groups, two experimental groups each sisting of monkeys and a trol group sisting of six monkeys. One of the experimental groups received immunogens taining single sensus gene inserts and the other received immunogens taining a cocktail of four complementary mosaic gene inserts. On weeks, and, the experimental groups of monkeys received priming immunizations by the intramuscular route with either a total of mg of M sensus gag or mosaic gag and a total of either mg of M sensus nef or mosaic nef plasmid DNA for each immunization. At week 33, we boosted the monkeys by simultaneous intramuscular and intradermal inoculations with a total of plaque-forming units of a recombinant vaccinia virus expressing the same antigens. We immunized the trol monkeys with empty vectors. Pooled peptide and peptide matrix interferon- enzyme-linked immunospot assays. We coated multiscreen 96-well plates overnight with 1 µl per well of µg ml 1 antibody to human IFN-γ (B27; BD Pharmingen) in endotoxin-free Dulbecco s-pbs (D-PBS). We then washed the plates three times with D-PBS taining.1% Tween-2 and blocked them for 2 h with RPMI- 16 taining 1% FBS. Then we added peptide pools and 2 1 PBLs to each well in 1-µl reaction volumes in triplicate for pooled peptides assays and in duplicate for peptide matrix assays. Each peptide pool was comprised of amino acid peptides overlapping by 11 amino acids. The pools covered the entire HIV-1 and proteins. Each peptide in a pool was present at a 1 µg ml 1 centration. After an 1-h incubation at 37 C, we washed the plates nine times with D-PBS taining.1% Tween-2 and once with distilled water. We then incubated the plates with 2 µg ml 1 biotinylated rabbit antibody to human IFN-γ (U-Cytech) for 2 h at 2 C, washed them six times with D-PBS taining.1% Tween-2 and incubated them for 2. h with a 1 in dilution of streptavidin-ap (Southern Biotechnology Associates). After five washes with D-PBS taining.1% Tween-2 and three washes with D-PBS, we developed the plates with NBT/BCIP chromogen (Pierce), stopped the reaction by washing with tap water, air dried the plates and read them with an ELISPOT reader (Cellular Technology Ltd.). We calculated the number of spot-forming cells per PBLs. The responses to the indicator peptides in the trol group of six monkeys were in the range of 1 SFCs per million PBLs. These values were always less than four times below the medium-alone values. CD + T lymphocyte depletion. We incubated PBLs from the monkeys with phycoerythrin-labeled CD-specific antibody and then with magnetic beads coated in antibody to phycoerythrin (Miltenyi Biotech) following manufacturer s protocol. We then sorted the labeled PBLs with a Miltenyi AutoMACS cell sorter to deplete CD + T lymphocytes. The efficiency of CD + T cell depletion in this study was 9.3% 99.7%. We measured IFN-γ ELISPOT responses in unfractionated and CD + T lymphocyte depleted PBLs from these monkeys. In the evaluation of the cellular immune response, if greater than one half of a SFC response by unfractionated PBLs was eliminated by CD + T lymphocyte depletion, we designated that response as CD + T lymphocyte mediated. The responses of CD + T lymphocyte depleted PBLs were designated as CD + T lymphocyte responses. These highly stringent criteria may underestimate the magnitude of the vaccine-elicited CD + T lymphocyte responses. Additional methods. Detailed methodology is described in the Supplementary Methods. doi:1.13/nm.21 nature medicine