Multi-low-dose mucosal simian immunodeficiency virus SIVmac239 challenge of cynomolgus macaques immunized with "hyperattenuated" SIV constructs.

Hyperattenuated simian immunodeficiency virus SIVmac239-derived constructs Delta5-CMV and Delta6-CCI are an effort to render SIV incapable of, in practical terms, both reversion and recombination while maintaining the immune features of SIV as a retrovirus. Primary inoculation of cynomolgus macaques with 10(8) 50% tissue culture infective doses (TCID(50)) of Delta5-CMV or Delta6-CCI induced low-level humoral and cellular responses detectable in the absence of measureable in vivo replication. The first of three DNA boosts resulted in elevated gamma interferon (IFN-gamma) enzyme-linked immunospot (ELISPOT) responses to Gag, Pol, and Env in the Delta5-CMV vaccine group compared to the Delta6-CCI vaccine group (P = 0.001). Weekly intrarectal challenge with a low dose of SIVmac239 followed by a dose escalation was conducted until all animals became infected. The mean peak viral load of the Delta5-CMV-vaccinated animals (3.7 x 10(5) copies/ml) was approximately 1 log unit lower than that of the control animals. More dramatically, the viral load set point of these animals was decreased by 3 log units compared to that of the controls (<50 versus 1.64 x 10(4) copies/ml; P < 0.0001). Seventy-five percent (6/8) of vaccine recipients controlled virus below 1,000 copies/ml for at least 6 months, with a subset controlling virus and maintaining substantial CD4 T-cell counts for close to 2 years of follow-up. The correlates of protection from SIV disease progression may lie in the rapidity and protective value of immune responses that occur early in primary SIV infection. Prior immunization with hyperattenuated SIVmac239, even if sterilizing immunity is not achieved, may allow a more advantageous host response.

Conflicting selective forces affect T cell receptor contacts in an immunodominant human immunodeficiency virus epitope.

Cytotoxic T lymphocytes (CTLs) are critical for the control of human immunodeficiency virus, but containment of virus replication can be undermined by mutations in CTL epitopes that lead to virus escape. We analyzed the evolution in vivo of an immunodominant, HLA-A2-restricted CTL epitope and found two principal, diametrically opposed evolutionary pathways that exclusively affect T cell-receptor contact residues. One pathway was characterized by acquisition of CTL escape mutations and the other by selection for wild-type amino acids. The pattern of CTL responses to epitope variants shaped which variant(s) prevailed in the virus population. The pathways notably influenced the amount of plasma virus, as patients with efficient CTL selection had lower plasma viral loads than did patients without efficient selection. Thus, viral escape from CTL responses does not necessarily correlate with disease progression.

Novel simian immunodeficiency virus CTL epitopes restricted by MHC class I molecule Mamu-B*01 are highly conserved for long term in DNA/MVA-vaccinated, SHIV-challenged rhesus macaques.

Simian immunodeficiency virus (SIV) infection of rhesus macaques provides an excellent model for investigating the basis of protective immunity against human immunodeficiency virus (HIV). One limitation of this model, however, has been the availability of a small number of known MHC class I-restricted CTL epitopes for investigating virus-specific immune responses. We assessed CTL responses against SIV Gag in a cohort of DNA/modified vaccinia virus Ankara (MVA)-vaccinated/simian-human immunodeficiency virus (SHIV)-challenged rhesus macaques. Here, we report the identification of five novel SIV CTL epitopes in Gag for the first time (Gag(39-46) NELDRFGL, Gag(169-177) EVVPGFQAL, Gag(198-206) AAMQIIRDI, Gag(257-265) IPVGNIYRR and Gag(296-305) SYVDRFYKSL) that are restricted by the common MHC class I molecule Mamu-B*01. CTL responses to these epitopes were readily detected in cryopreserved PBMC in multiple animals up to 62 weeks post-infection, both by IFN-gamma enzyme-linked immunospot assay and intracellular IFN-gamma staining. Importantly, viral sequencing results revealed that these epitopes are highly conserved in the SIV-challenged macaques over a long period of time, indicating functional constraints in these regions. Moreover, the presence of CTL responses targeting these epitopes has been confirmed in two independent cohorts of rhesus macaques that have been challenged by SHIV or SIV. Our findings provide valuable candidates for poly-epitope vaccines and for long-term quantitative monitoring of epitope-specific CD8(+) responses in the context of this common Mamu class I allele. It may thus help increase the supply of rhesus macaques in which epitope-specific immunity can be studied in the context of SIV vaccine design.

Conformational constraints imposed on a pan-neutralizing HIV-1 antibody epitope result in increased antigenicity but not neutralizing response.

2F5 is one of the few broadly neutralizing monoclonal antibodies against type 1 Human Immunodeficiency Virus (HIV-1). It recognizes the amino acid sequence ELDKWAS in gp41. We have previously identified a number of immunotargeting 2F5-reactive candidate immunogens. Three of them (designated H-BT1-3) have the ELDKWAS sequence constrained at beta-turn sites within the immunoglobulin heavy chain. Two others (L-CT and L-CTx3) have the sequence attached at the C-terminus of the immunoglobulin light chain with minimal conformational constraints. In the present investigation, the H-BTs were found to bind 2F5 with up to 10-fold higher affinities than their unconstrained counterpart. When used as immunogens, immunogen-specific antibodies were induced with or without adjuvant, confirming the immunotargeting potential of these immunogen constructs. While HIV-1 gp160 cross-reactive antibodies were induced, virus neutralization was not detected. Thus, factors other than 2F5 binding affinity may have a critical role to play in the design of a 2F5-based vaccine.

Sequence of beta(2)-microglobulin from rhesus macaque ( Macaca mulatta) includes an allelic variation in the 3'-untranslated region.

The rhesus macaque ( Macaca mulatta) has become a popular animal model for several human infectious diseases, such as HIV (modeled by SIV infection), hepatitis, and malaria. Investigation of T-cell responses in experimental infectious diseases in rhesus macaques has benefited from an expanding understanding of the diversity of macaque MHC class I heavy chains and the restriction of antigen presentation by macaque class I molecules. Here we add to this understanding with the first nucleotide sequences of M. mulatta beta(2)-microglobulin (beta(2)m) mRNA, including a portion of the 3'-untranslated region (3'UTR). In pairwise comparison, the beta(2)m protein of M. mulatta differs from human and chimpanzee beta(2)m by nine amino-acid substitutions (92% identity), and from Macaca fascicularis by one amino-acid difference in the signal peptide region (99% identity). Allelic variations were identified at one site in the 3'UTR. A structural analysis of human or chimpanzee beta(2)m and M. mulatta beta(2)m suggests that the differences cluster in three solvent-exposed clusters and do not involve contacts with the class I heavy chain. We predict that human and macaque beta(2)m should bind interchangeably with the class I heavy chains of the other species, and show that four M. mulatta class I alleles form cell surface complexes with human beta(2)m. Further, we predict that W6/32 (a monoclonal antibody that recognizes a combined epitope of some class I heavy chains and beta(2)m with a subtle species dependence) should bind similarly human or macaque class I molecules that are bound with beta(2)m of either species, supported by evidence of recognition of both heterologous and homologous complexes of macaque class I heavy chains. Our findings contribute to the growing understanding of rhesus macaque histocompatibility antigens and antigen presentation, and to the phylogeny of beta(2)m in primates.

Assessing human alloimmunization as a strategy for inducing HIV type 1 neutralizing anti-HLA responses.

Xenovaccination of rhesus macaques with human HLA Class I and II proteins has been demonstrated to elicit protective immunity against challenge with SIV grown in human cells. To determine if alloimmunization in humans could lead to protective immunity against HIV-1, we prospectively followed a small group of women receiving whole-cell alloimmunization in the form of leukocyte immunotherapy for recurrent spontaneous abortion. Whole-cell vaccine recipients and their respective partners (referred to as donors) provided pre- and postimmune blood samples for analysis. Study participants were HLA typed by sequence-specific PCR and antibodies specific for HLA Class I and II antigens were measured in recipient plasma. To determine if anti-HLA antibody responses detected in recipient plasma samples were capable of neutralizing HIV-1 in vitro, we grew laboratory strain HIV-1(IIIB) and primary isolate HIV-1(301660) in donor-derived CD4(+) T lymphocytes. The ability of purified whole IgG from responding patients to neutralizing infectivity of the respective donor-derived virus was then assayed in vitro. All donor-recipient pairs were determined to be HLA discordant for at least one Class I and one Class II locus. Two of seven female recipients in total made strong anti-HLA antibody responses specific to the HLA haplotype of the male donor in response to the alloimmunization regimen. For one recipient, IgG antibodies specific for donor HLA Class I and II antigens were able to neutralize both HIV-1(IIIB) and a primary isolate HIV-1(301660). In addition polyclonal anti-HLA class II antibodies against a single determinant (DR4) of this donor were also neutralizing. In contrast, the other recipient exhibiting antibodies only against donor HLA Class I antigens did not neutralize HIV-1(IIIB). Using samples from a small number of women undergoing leukocyte immunotherapy, we have demonstrated for the first time that allele-specific anti-HLA antibodies elicited through human alloimmunization are capable of neutralizing HIV-1 in vitro.