Cross-protection in NYVAC-HIV-1-immunized/HIV-2-challenged but not in NYVAC-HIV-2-immunized/SHIV-challenged rhesus macaques.

OBJECTIVES: Immunization with attenuated poxvirus-HIV-1 recombinants followed by protein boosting had protected four of eight rhesus macaques from HIV-2SBL6669 challenge. The present study was designed to confirm this result and to conduct the reciprocal cross-protection experiment. METHODS: Twenty-four macaques were primed with NYVAC (a genetically attenuated Copenhagen vaccinia strain) recombinants with HIV-1 and HIV-2 env and gag-pol or NYVAC vector alone and boosted with homologous, oligomeric gp160 proteins or adjuvant only. Binding and neutralizing antibodies, cytotoxic T-lymphocytes (CTL) and CD8 T cell antiviral activity (CD8AA) were evaluated. One half of each immunization and control group were intravenously challenged with SHIV(HXB2) the other half was challenged with HIV-2SBL6669,. Protective outcome was assessed by monitoring virus isolation, proviral DNA and plasma viral RNA. RESULTS: Both immunization groups developed homologous binding antibodies; however, homologous neutralizing antibodies were only observed in NYVAC-HIV-2-immunized macaques. While no cross-reactive neutralizing antibodies were detected, both immunization groups displayed cross-reactive CTL. Significant CD8AA was observed for only one NYVAC-HIV-2-immunized macaque. Virological assessments verified that both NYVAC-HIV-1 and NYVAC-HIV-2 immunization significantly reduced viral burdens and partially protected against HIV-2 challenge, although cross-protection was not at the level that had been previously reported. Humoral antibody and/or CTL and CD8AA were associated with protection against homologous HIV-2 challenge, while cellular immune responses seemed more important for cross-protection. No significant protection was observed in the SHIV-challenged macaques, although NYVAC-HIV-1 immunization resulted in significantly lower viral burdens compared with controls. CONCLUSIONS: Further delineation of cross-reactive mechanisms may aid in the development of a broadly protective vaccine.

Targeting the mucosa: genetically engineered vaccines and mucosal immune responses.

The discovery that inoculation of DNA leads to strong and long lasting immune responses generated enthusiasm to assess the efficacy of various genetically engineered vaccines against mucosally acquired infections. Various techniques have been used to generate the most suitable DNA vaccines, ranging from immunization with naked DNA to utilizing genetically engineered recombinant viruses and bacteria to deliver the DNA. Different DNA vaccine modalities and mucosal immune responses to them have been discussed. It has been shown that even though intramuscular and intradermal immunization with these vaccines generates strong systemic responses, mucosal responses are not induced. It has been proposed that the site of immunization determines mucosal immune responses and that primed lymphocytes preferentially accumulate at sites where they have been induced thus generating the strongest cellular and antibody responses at the site of vaccination. The impact of the site of induction on mucosal immune responses to vaccines is discussed. It is possible to enhance desired vaccine effects in the mucosa and to modify the undesirable side effects. Cytokines such as IL-2, IL-12, IL-15 and IL-18 have been used to enhance CTL activity while IL-5, IL-6 and the chemokine MIP-1 alpha have shown the capacity to increase IgA responses to vaccines.

A comprehensive panel of near-full-length clones and reference sequences for non-subtype B isolates of human immunodeficiency virus type 1.

Non-subtype B viruses cause the vast majority of new human immunodeficiency virus type 1 (HIV-1) infections worldwide and are thus the major focus of international vaccine efforts. Although their geographic dissemination is carefully monitored, their immunogenic and biological properties remain largely unknown, in part because well-characterized virological reference reagents are lacking. In particular, full-length clones and sequences are rare, since subtype classification is frequently based on small PCR-derived viral fragments. There are only five proviral clones available for viruses other than subtype B, and these represent only 3 of the 10 proposed (group M) sequence subtypes. This lack of reference sequences also confounds the identification and analysis of mosaic (recombinant) genomes, which appear to be arising with increasing frequency in areas where multiple sequence subtypes cocirculate. To generate a more representative panel of non-subtype B reference reagents, we have cloned (by long PCR or lambda phage techniques) and sequenced 10 near-full-length HIV-1 genomes (lacking less than 80 bp of long terminal repeat sequences) from primary isolates collected at major epicenters of the global AIDS pandemic. Detailed phylogenetic analyses identified six that represented nonrecombinant members of HIV-1 subtypes A (92UG037.1), C (92BR025. 8), D (84ZR085.1 and 94UG114.1), F (93BR020.1), and H (90CF056.1), the last two comprising the first full-length examples of these subtypes. Four others were found to be complex mosaics of subtypes A and C (92RW009.6), A and G (92NG083.2 and 92NG003.1), and B and F (93BR029.4), again emphasizing the impact of intersubtype recombination on global HIV-1 diversification. Although a number of clones had frameshift mutations or translational stop codons in major open reading frames, all the genomes contained a complete set of genes and three had intact genomic organizations without inactivating mutations. Reconstruction of one of these (94UG114.1) yielded replication-competent virus that grew to high titers in normal donor peripheral blood mononuclear cell cultures. This panel of non-subtype B reference genomes should prove valuable for structure-function studies of genetically diverse viral gene products, the generation of subtype-specific immunological reagents, and the production of DNA- and protein-based subunit vaccines directed against a broader spectrum of viruses.

HIV-1 recombinant poxvirus vaccine induces cross-protection against HIV-2 challenge in rhesus macaques.

Rhesus macaques were immunized with attenuated vaccinia or canarypox human immunodeficiency virus type 1 (HIV-1) recombinants and boosted with HIV-1 protein subunits formulated in alum. Following challenge with HIV-2SBL6669, three out of eight immunized macaques resisted infection for six months and another exhibited significantly delayed infection, whereas all three naive controls became infected. Immunizations elicited both humoral and cellular immune responses; however, no clear correlates of protection were discerned. Although more extensive studies are now called for, this first demonstration of cross-protection between HIV-1 and -2 suggests that viral variability may not be an insurmountable problem in the design of a global AIDS vaccine.

Humoral and cellular immune responses in rhesus macaques infected with human immunodeficiency virus type 2.

Eighteen rhesus macaques were inoculated with either an infectious molecularly cloned human immunodeficiency virus type 2 (HIV-2)SBL/ISY, or with one of eight mutants defective in one or more accessory genes. The immune responses generated by the macaques were monitored for up to 2 years postinfection. All the macaques except those that received mutants lacking the vpr or vif genes demonstrated low to moderate antibody titers. Macaques inoculated with vpx- mutants exhibited a persistent serological response, suggesting continuous virus expression even in the absence of detectable virus in the peripheral blood mononuclear cells (PBMCs). Neutralizing antibodies developed in only four macaques. In general, low-level cytotoxic T lymphocyte (CTL) activity, not clearly HIV-2 specific, was detected in PBMCs. However, one virus-negative macaque exhibited significant HIV-2-specific CTL activity in an enriched CD8+ cell population from PBMCs, suggesting clearance of the viral infection. In addition, CTL activity against the Env and Gag/Pol epitopes of HIV-2 by CD8+ lymphocytes from the spleens and lymph nodes of two infected macaques, in one case requiring CD8+ T cell enrichment and in the other clearly evident in unfractionated tissue lymphocytes, was demonstrated for the first time. This sequestration of tissue CTLs occurred in the absence of significant levels of circulating CTLs in the blood. Our results suggest that routine monitoring of PBMCs may sometimes be inadequate for detecting cell-mediated immune responses. Elucidation of immune correlates of vaccine protection may therefore require sampling of lymphoid tissues and assessment of enriched CD8+ populations.

Subgroup G HIV type 1 isolates from Nigeria.

PIP: Nucleotide sequences were obtained for portions of the envelope and gag genes of 4 HIV-1 isolates from Nigeria. The gag gene sequences clustered with previously described gag sequences from Gabon, Zaire, and Taiwan, which form the G clade of HIV-1. This documented for the first time the presence of subgroup G viruses in Nigeria. The env gene sequences were most closely related to env sequences reported from 2 isolates previously classified as gag subgroup G and, with those 2 env sequences, defined the subgroup G env genotype. Virus isolates were obtained by cocultivation of peripheral blood mononuclear cells (PBMCs) from Nigerian AIDS patients (isolates JV1018 and JP88) or healthy prostitutes (G3 and G9) with normal uninfected PBMCs (JV1083, JP88, and G3) or GEM-SS, a T cell line (G9). Clones containing env and gag gene sequences were obtained by polymerase chain reaction amplification, using DNA from these cultures. The region of gag amplified by primers SK37 and SK39 containing BamHI and KpnI sites, respectively, in 5' tails, was cloned into the KpnI-BamHI site of pKSBluescript and sequenced. When the gag sequences from 2 of the Nigerian viruses were subtyped by weighted parsimony, both viruses grouped with others of the gag G subgroup. A similar analysis of env sequences from all 4 Nigerian isolates showed that they were most closely related to 2 env sequences that had been assigned to env subgroup G. The Nigerian env sequences clustered more closely with each other than they did with the other 2 subgroup G viruses. Consensus of the inferred amino acid sequences of the 4 env genes showed that the V3 loop closely resembled the A to F consensus. 8 positions in the Nigerian consensus sequence appeared to be unique. However, whether these are signature residues for Nigerian subgroup G isolates or for subgroup G isolates will require the characterization of more isolates.^ieng

Comparison of different vaccines and induced immune response against Campylobacter jejuni colonization in the infant mouse.

The degree of protection conferred by vaccinated dams on infant mice against colonization by Campylobacter jejuni depended on the bacterial strain, preparation, and route of administration of the vaccine. In some instances of homologous protection, serum bactericidal titres correlated well with protection. However, boiled C. jejuni vaccine, which was non-protective, also elicited a strong bactericidal antibody response. Conversely, bactericidal activity could not be demonstrated against strains capable of cross-protection. There was a good correlation between high campylobacter-specific IgG response and bactericidal activity.

Cross-protection of infant mice against intestinal colonisation by Campylobacter jejuni: importance of heat-labile serotyping (Lior) antigens.

An association of the heat-labile antigens detected by the Lior serotyping scheme with ability to protect infant mice against gastrointestinal colonisation with Campylobacter jejuni has been established. Overall, 39 (57%) of 68 infant mice challenged with a heterologous strain of the same Lior serotype as the vaccine strain were protected, compared with 40 (85%) of 47 infants protected against a homologous challenge. In contrast, none of the infant mice challenged with a strain carrying the same heat-stable antigens (i.e., of the same Penner serotype as the vaccine strain) were protected.

The mechanism of protection of infant mice from intestinal colonisation with Campylobacter jejuni.

BALB/c mice, vaccinated intraperitoneally with a heat-killed (62 degrees C) suspension of Campylobacter jejuni before mating, completely protect c. 90% of their own infants from intestinal colonisation. This protection has now been investigated further in fostering experiments. Fostering by vaccinated dams within the first 24 h of life prevented intestinal colonisation in 50% of infants from non-vaccinated dams, and reduced colonisation in a further 25%. Infants from vaccinated dams, even if allowed to receive their own mothers' colostrum and milk, became susceptible to challenge when subsequently fostered by non-vaccinated dams. Immunity in experimentally infected infant mice depended upon the consumption of immune milk at and after the time of challenge. High concentrations of IgG antibodies specific for C. jejuni were found in the serum and mammary secretion of vaccinated dams, but there was very little specific IgA antibody.