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

Abstract

An effective HIV vaccine must elicit immune responses that recognize genetically diverse viruses. It must generate CD8+ T lymphocytes that control HIV replication and CD4+ T lymphocytes that provide help for the generation and maintenance of both cellular and humoral immune responses against the virus. 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. 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 constructs expressing either consensus or polyvalent mosaic proteins. As compared to consensus immunogens, the mosaic immunogens elicited CD8+ T lymphocyte responses to more epitopes of each viral protein than did the consensus immunogens and to more variant sequences of CD8+ T lymphocyte epitopes. This increased breadth and depth of epitope recognition may contribute both to protection against infection by genetically diverse viruses and to the control of variant viruses that emerge as they mutate away from recognition by cytotoxic T lymphocytes.

Fig. 1

Number of epitope-specific responses by CD4+ and CD8+ T lymphocytes of vaccinated monkeys. (a) CD4+ and CD8+ T lymphocyte responses from 7 monkeys per group were assessed for their recognition of specific epitopes of each of the 10 indicator Gag and Nef proteins in IFN-γ ELISpot assays. Unfractionated and CD8+ lymphocyte-depleted PBL were assessed for their recognition of specific epitope peptides. Each animal’s responses to each peptide series are shown in different colored bars: clade A (aqua), clade B (red), clade C (purple), and clade G (blue). (b) The breadth of CD4+ and CD8+T lymphocyte responses by individual monkeys was determined as follows: if a 15-mer peptide from one of the 10 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 different indicator proteins were recognized by a T lymphocyte population, they were also scored as one positive response. For the Mosaic-vaccinated monkeys, epitopes in the Gag protein that were recognized are shown in blue bars and epitopes in the Nef protein that were recognized are shown in aqua bars. For the Consensus-vaccinated monkeys, recognized Gag and Nef epitopes are shown in green and light green bars, respectively. (c) The depth of CD4+ and CD8+T lymphocyte responses by individual monkeys was determined by counting the responses made to all variant peptides from each epitopic region of either the Gag or the Nef protein.

Fig. 2

Examples of the depth of vaccine-elicited CD8+ T lymphocyte responses. Consensus and mosaic vaccine-induced CD8+ T lymphocyte responses were assessed for recognition of variant forms of the same region of a viral protein. For each example shown, the variant HIV-1 sequences are displayed aligned to the M group consensus of that sequence. Amino acid identity to the consensus sequence is shown 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 CD8+ T lymphocyte response: CD8+ T lymphocytes from monkey 58 recognize only the peptide sequence that matches the vaccine sequence. (b) A cross-reactive CD8+ T lymphocyte response: three different variants of peptide 15 and 16 sequences exist in 10 indicator gag proteins, and CD8+ T lymphocytes from monkey 228 recognize all three variants. (c) A highly restricted CD8+ T lymphocyte response: CD8+ T lymphocytes of monkey 65 recognize only the variant peptide that matches one of the four mosaic sequences used in the mosaic immunogen cocktail. (d) A cross-reactive CD8+ T lymphocyte response: CD8+ T lymphocytes from monkey 65 recognize four different variant forms of the peptide, all of which differ in sequence from the vaccine immunogens.

Fig. 3

Breadth and depth of Gag- and Nef-specific CD8+ T lymphocyte responses in 7 individual monkeys from each vaccine group. All of the Gag and Nef peptides recognized by PBLs of individual monkeys were aligned to the HIV-1 M group consensus sequences as described in the legend of Figure 2. Lymphocyte recognition of any peptide sequence is shown in rectangles of a different shade of green, orange, or yellow and non-recognition of a peptide is shown in unshaded rectangles.

Fig. 4

Cross-clade epitope recognition by CD4+ and CD8+ T lymphocytes of 7 individual monkeys from each vaccine group. The stacks of 10 rectangles represent the 10 sets of indicator peptides with A1 and A2 shown in turquoise; B1 and B2 in red; C1, C2, C3 and C4 in purple; and G1 and G2 in dark blue. For each monkey, in the left hand column (min1) if at least one peptide from an indicator peptide set was recognized by either (a) CD4+ or (b) CD8+ T lymphocytes, the rectangle is shaded with its representative color. If no PBL responses to a particular set of indicator peptides are detected, the rectangle is shown unshaded. Recognition of one, two or three epitopes per strain of indicator proteins by CD4+ and CD8+ T lymphocytes of each of the 7 monkeys in both vaccine groups are shown as min1, min 2 and min 3, respectively.