The V3 region of the human immunodeficiency virus type 1 gp120 Env protein is a key domain in Env due to its role in interacting with the coreceptors CCR5 and CXCR4. We examined potential subtype-specific V3 region differences by comparing patterns of amino acid variability and probing for subtype-specific structures using 11 anti-V3 monoclonal antibodies (V3 MAbs). Differences between the subtypes in patterns of variability were most evident in the stem and turn regions of V3 (positions 9 to 24), with the two subtypes being very similar in the base region. The characteristics of the binding of V3 MAbs to Env proteins of the subtype B virus JR-FL and the subtype C virus BR025 suggested three patterns, as each group of MAbs recognized a specific conformation-or sequence-based epitope. Viruses pseudotyped with Env from JR-FL and BR025 were resistant to neutralization by the V3 MAbs, although the replacement of the Env V3 region of the SF162 virus with the JR-FL V3 created a pseudotyped virus that was hypersensitive to neutralization. A single mutation in V3 (H13R) made this chimeric Env selectively resistant to one group of V3 MAbs, consistent with the mAb binding properties. We hypothesize that there are intrinsic differences in V3 conformation between subtype B and subtype C that are localized to the stem and turn regions and that these differences have two important biological consequences: first, subtype B and subtype C V3 regions can have subtype-specific epitopes that will inherently limit antibody cross-reactivity, and second, V3 conformational differences may potentiate the frequent evolution of R5-into X4-tropic variants of subtype B but limit subtype C virus from using the same mechanism to evolve X4-tropic variants as efficiently.The binding of the human immunodeficiency virus type 1 (HIV-1) to its target cell is mediated by the viral envelope glycoprotein Env. The glycoprotein is expressed as a gp160 precursor that is proteolytically cleaved into two mature subunits, the gp120 surface protein and the gp41 transmembrane protein. On the surface of the virus, Env is present as a trimer of the gp120 and gp41 heterodimers (2, 62). When the virus binds to its primary receptor, CD4, Env undergoes conformational changes, exposing previously hidden regions and creating new structural elements. Env then binds to a coreceptor, usually the chemokine receptor CCR5 or CXCR4 (23, 26, 80), which triggers further structural changes that promote fusion to the target cell (27, 35).The Env sequence can be viewed as being composed of regions that are relatively constant (C1 to C5) and regions that on a population basis are much more variable (V1 to V5) (70). As the only viral protein expressed on the surface of the virus, Env is the sole target of neutralizing antibodies that supply selective pressure to favor mutated variants capable of evading the immune response (47, 79). The V3 region is associated with coreceptor preference and physically contacts the chemokine receptor as part of the fusion process (23,26,80). Sequen...

Recently, a vaccine consisting of DNA priming followed by boosting with modified vaccinia Ankara (MVA) has provided long-term protection of rhesus macaques against a virulent challenge with a chimera of simian and human immunodeficiency viruses. Here, we report studies on the development of the DNA component for a DNA/MVA HIV vaccine for humans. Specifically, we assess the ability of a codon-optimized Gag-expressing DNA and two noncodon-optimized Gag-Pol-Env-expressing DNAs to prime the MVA booster dose. The codon-optimized DNA expressed virus-like particles (VLPs), whereas one of the noncodon-optimized DNAs expressed VLPs and the other expressed aggregates of HIV proteins. The MVA boost expressed Gag-Pol and Env and produced VLPs. Immunogenicity studies in macaques used one intramuscular prime with 600 microg of DNA and two intramuscular boosts with 1 x 10(8) pfu of MVA at weeks 8 and 30. The codon-optimized and noncodon-optimized DNAs proved similar in their ability to prime anti-Gag T cell responses. The aggregate and VLP-expressing Gag-Pol-Env DNAs also showed no significant differences in their ability to prime anti-Env Ab responses. The second MVA booster dose did not increase the peak CD4 and CD8 T cell responses, but increased anti-Env Ab titers by 40- to 90-fold. MVA-only immunizations elicited 10-100 times lower frequencies of T cells and 2-4 lower titers of anti-Env Ab than the Gag-Pol-Env DNA/MVA immunizations. Based on the breadth of the T cell response and a trend toward higher titers of anti-Env Ab, we are moving forward with human trials of the noncodon-optimized VLP-expressing DNA.

Recently, a simian/human immunodeficiency virus (SHIV) vaccine consisting of priming with a Gag-Pol-Env-expressing DNA and boosting with a Gag-Pol-Env-expressing recombinant modified vaccinia Ankara (rMVA) has successfully controlled a virulent SHIV challenge in a macaque model. In this, and the accompanying paper, we report on the construction and testing of a Gag-Pol-Env DNA/MVA vaccine for HIV-1/AIDS. The DNA vaccine, pGA2/JS2, expresses aggregates of Gag proteins and includes safety mutations that render it integration, reverse transcription, and packaging defective. The rMVA vaccine, MVA/HIV 48, is integration and reverse transcription defective and has a truncated Env to enhance expression on the plasma membrane. In a study in rhesus macaques, priming with pGA2/JS2 and boosting with MVA/HIV 48 raised high frequencies of T cells for Gag and Env and lower frequencies of T cells for PR, RT, and Tat. Stimulations with five peptide pools for Gag and seven peptide pools for Env revealed epitopes for cellular immune responses throughout Gag and Env. On average, CD4 T cells from the vaccinated animals recognized 7.1 peptide pools and CD8 T cells, 3.2 peptide pools. Both the height and the breadth of the elicited cellular response provide hope that this multiprotein DNA/MVA vaccine will successfully control clade B isolates of HIV-1, as well as contribute to the control of other clades and recombinant forms of HIV-1/AIDS.

Here, we evaluate the T cell responses raised by our HIV-1 clade B DNA/MVA vaccine for recognition of a HIV-1 circulating recombinant form (CRF) AG Gag sequence (CRF-02). The cross-clade activity for the AG sequence was better conserved for CD8 than CD4 T cells. CD8 T cells exhibited 75% conservation for height and 83% conservation for breadth, whereas CD4 responses exhibited 45% conservation for height and 50% conservation for breadth. Five CD8 epitopes and 8 CD4 epitopes were mapped. Three of the 5 CD8 epitopes and 2 of the 8 CD4 epitopes were conserved across multiple HIV-1 clades. Impressively, all of the CD8 epitopes and half of the CD4 epitopes have been reported for human infections. Our results demonstrate that the clade B DNA/MVA HIV vaccine elicits T cell responses against epitopes that are conserved in multiple clades and recognized by humans and macaques.