Enhanced cellular immunity and systemic control of SHIV infection by combined parenteral and mucosal administration of a DNA prime MVA boost vaccine regimen.

The immunogenicity and protective efficacy of a DNA and recombinant modified vaccinia Ankara (MVA) vaccine administered by two different routes were investigated. DNA expressing HIV-1 IIIB env, gag, RT, rev, tat and nef, and MVA expressing HIV-1 IIIB nef, tat and rev and simian immunodeficiency virus (SIV) macJ5 gag/pol and vaccinia HIV-1 env, were used as immunogens. Four cynomolgus macaques received DNA intramuscularly (i.m.) at month 0 and intrarectally (i.r.) and intra-orally (i.o.) at 2 months, followed by MVA i.m. at 4 months and i.r. and i.o. at 8 months. Another group of four monkeys received the same immunogens but only i.m. Overall, stronger cellular immune responses measured by ELISPOT and T-cell proliferation assay were detected in the group primed i.m. and boosted mucosally. Following homologous intravenous simian-human immunodeficiency virus (SHIV) challenge, one of eight vaccinated animals was completely protected. This monkey, immunized i.m. and i.r.+i.o., exhibited the highest levels of HIV Env, Nef and Tat antibodies, high HIV Tat cytotoxic T-lymphocyte activity and T-lymphocyte proliferative responses to HIV Env. Four weeks post-challenge none of the monkeys immunized i.m. and i.r.+i.o., and only two out of four animals immunized i.m., demonstrated detectable plasma viral RNA levels. In contrast, all eight control animals had demonstrable plasma viral RNA levels 4 weeks post-challenge. Thus, stronger cellular immune responses and reduction of challenge virus burden were demonstrated in animals immunized i.m. as well as mucosally, compared with animals immunized i.m. only. The breadth and magnitude of the induced immune responses correlated with protective efficacy.

Induction of immune responses and break of tolerance by DNA against the HIV-1 coreceptor CCR5 but no protection from SIVsm challenge.

An inactivating mutation in the human CCR5 gene reduces the risk of HIV-1 infection in individuals with homozygous alleles. We explored whether genetic immunization would induce an immune response directed to CCR5 structures and if immunological tolerance toward endogenous CCR5 could be broken. We also studied whether this immunization approach could protect cynomolgus monkeys from an infection, with SIVsm, which primarily uses CCR5 as a coreceptor. Epidermal but not intramuscular delivery of the CCR5 gene to mice elicited strong IgG antibody binding responses to CCR5. Intramucosal immunization of cynomolgus macaques with CCR5 DNA followed by boosts with CCR5 peptides induced prominent IgG and IgA antibody responses in serum and vaginal washings. The CCR5-specific antibodies neutralized the infectivity of primary human R5 HIV-1 strains, and the macaque SIVsm but not that of a tissue culture-adapted X4 HIV-1 strain. The consecutive CCR5 gene and CCR5 peptide immunizations induced B- and T-cell responses to peptides representing both human and macaque amino acid sequences of the respective CCR5 proteins. This indicates that tolerance was broken against endogenous macaque CCR5, which has a 98% homology to the human CCR5 gene. After the final boost, the vaccinated monkeys together with two control monkeys were challenged with SIVsm. Neither protection against nor enhancement of SIVsm infection was achieved.

DNA-plasmids of HIV-1 induce systemic and mucosal immune responses.

DNA-based immunization has been shown to induce protective immunity against several microbial pathogens including HIV-1. Several routes of DNA vaccination have been exploited. However, the properties of the immune responses seem to differ with the different routes used for DNA delivery, ultimately affecting the outcome of experimental challenge. We measured the primary immune response following one vaccination. This report presents differences associated with three different DNA delivery routes: intramuscular injection, intranasal application, and gene-gun based immunization. Induction of systemic humoral immune responses was achieved most efficiently by either intranasal or gene-gun mediated immunization, followed by intramuscular injection. Mucosal IgA was reproducibly induced by intranasal instillation of the DNA, and found in lung washings, faeces, and vaginal washings. Cytotoxic T cells were not induced by a single immunization, but were observed after three immunizations using intramuscular injections.

Induction of mucosal IgA by a novel jet delivery technique for HIV-1 DNA.

Novel ways of delivering plasmid DNA to elicit humoral IgA, IgG and cell-mediated immune responses in mice were investigated. Intraoral administration of DNA in the cheek, using a jet immunization technique, elicited the highest IgA mucosal responses. Intranasal immunization gave strong mucosal IgA responses and persistent systemic IgG. Immunoglobulin isotype analysis revealed an IgG1 profile for intramuscular tongue and gene gun immunizations and an IgG2a profile following oral jet injection and intranasal application. The route of delivery was of importance for the characteristics and quality of the mucosal immune response following DNA immunization. For DNA vaccine delivery, the intraoral jet injection technique has the advantages of being a simple and rapid way of administering the DNA in solution and of provoking specific mucosal IgA when administered in the mucosal associated lymphoid tissue.

Immunoglobulin G abnormalities in HIV-1 infected individuals with lymphoma.

BACKGROUND: Polyclonal B-cell activation precedes the occurrence of malignant B-cell clones. Several recent reports suggest a perturbed cytokine regulation in HIV-related lymphomagenesis and Epstein-Barr virus (EBV) involvement in approximately half of the cases with generalized lymphoma. OBJECTIVES: We investigated whether altered immunoglobulin properties would be detected by fine analysis of the immunoglobulin G (IgG) subclass patterns against HIV and EBV epitopes. STUDY DESIGN: HIV-1 infected patients in early stage, late stage and with lymphoma were analyzed by ELISA for anti HIV and EBV IgG class and subclass antibodies. Avidity and affinity of the antibodies were studied. The lymphoma patients were also studied by PCR for EBV DNA in serum. RESULTS: The total IgG reactivity to several HIV antigens was similar in the three patient groups. However, lymphoma patients had a more restricted subclass pattern with significantly lower IgG1 and IgG3 anti gp120 titers compared to other HIV-infected patients but good and persistent total IgG and IgG1 (excluding the gp120 antigen) reactivities in contradiction to their low CD4 counts. IgG4 reactivity was sparse, detectable to significant levels in the symptomatic group only. The observed relative affinity of the HIV-specific IgG and IgG1 of lymphoma patients was similar to that of asymptomatic and symptomatic patients. The subclass reactivity to the EBV peptide was similar in all groups but lymphoma patients with EBV DNA in serum exhibited significantly lower anti EBV peptide titers than those who were EBV DNA negative. CONCLUSION: These findings indicate that subclass analysis to defined viral antigens may be a means to detect immune dysregulation in tumor development.

Recognition of prominent viral epitopes induced by immunization with human immunodeficiency virus type 1 regulatory genes.

Mice immunized with the regulatory genes nef, rev, and tat from human immunodeficiency virus type 1 developed both humoral and cellular immune responses to the gene products Nef, Rev, and Tat. This study demonstrates that it is feasible to induce immune reactions to all of these regulatory gene products. Humoral responses were seen after DNA boosts, while potent T-cell proliferative responses were noted already after a single immunization. A Th1-directed immune response was demonstrated early after immunization. A 3- to 75-fold-stronger T-cell response was seen in animals receiving DNA epidermally compared to that in animals receiving intramuscular injections. Nef, Rev, and Tat putative B- and T-cell epitopes were clearly mapped by using peptides derived from the regulatory proteins and were similar to those which are detected in human immunodeficiency virus infection. Although immunization by the Nef, Rev, and Tat proteins raised high immunoglobulin G titers in serum, the epitope spreading appeared broader after DNA immunization. The combination of all of these regulatory genes together with two genes for structural proteins, the envelope and gag genes, demonstrated that a combined approach is feasible in that reactivities to all antigens persisted or were even augmented. No interference between plasmids was noted.

Nucleic acid vaccination with HIV regulatory genes: a combination of HIV-1 genes in separate plasmids induces strong immune responses.

The concept of combining several genes in order to immunize against a microbial agent has been tested. We selected human immunodeficiency virus (HIV) genes that individually have been shown to mediate immune responses against HIV proteins. These proteins were the regulating genes/proteins of HIV-1 rev, tat and nef as well as structural genes for gp160 under the control of rev, and the capsid p24 represented by the larger precursor gene p37. Two findings were of particular interest. The combination of these five gene constructs gave strong reactivity to all of them, compared with previous results using each one in single injections. The intranasal immunization route gave good mucosal reactivity by inducing IgG, IgA and T-cell proliferative responses.

Autoreactivity in HIV-infected individuals does not increase during vaccination with envelope rgp160.

The HIV-1 envelope protein contains several regions with amino acid homology to HLA class I and class II molecules. We evaluated possible changes in antibody responses to those regions during vaccination with rgp 160 produced in a baculovirus system. Forty asymptomatic HIV-infected patients with CD4 cell counts above 400 were vaccinated with rgp 160. Twenty-one patients were tissue-typed as HLA A2. Sixty-two percent of these patients exhibited cytotoxic lymphocyte antibodies directed to CD8+, HLA A2 cells. This cytotoxicity decreased during HIV gp160 vaccination. In order to further characterize the specificity of these responses, analogues of HLA class I and HLA-DR peptides were chemically synthesized together with their correct HIV-1 gp160 sequences. Enzyme-linked immunosorbent assays (ELISA) with sera from before, during and after immunization were performed with HIV proteins, peptides and their homologues. All patients showed an increase in their previously poor specific T-cell activation to gp160. Fourteen patients developed increased avidities or titres to HIV proteins and/or peptides. Contrarily, serum IgG titers to the HLA homologous peptides were initially low and decreased further during the course of vaccination. This decrease occurred in the majority of patients, 35-40 of the 40 individuals, depending on the antigen. Independent measures of autoantibodies to Ro/SS-A and La/SS-B remained undetectable.

Locations of antibody binding sites within conserved regions of the hepatitis C virus core protein.

The binding sites for human antibodies recognising antigenic domains within the hepatitis C virus (HCV) core protein were analyzed using synthetic peptides. Omission peptide analogues where a pair of residues was sequentially omitted were produced corresponding to the regions 1-18, 11-28, 21-38, 51-68, and 101-118. The N-terminal part of HCV core was found to contain three distinct antibody binding sites, which includes the previously reported one at residues 9-16. The other two were located at residues 19-26 and residues 29-34. Within the region 51-68 two overlapping sites were found, the first at residues 51-60 and the second at residues 59-68. Within the region 101-118, residues 107-114 were identified as the binding site, which contains two residues differing between genotypes I/II and III/VI. Thus the HCV core protein contains at least six distinct linear antibody binding sites, located at regions highly conserved between the genotypes of HCV. The human recognition of these regions show a variation with respect to the amino- and carboxy-terminal extension of each individual binding site. These findings will have implications for the prediction of the structure of the HCV core protein, since these antibody binding sequences are likely to be more or less accessible from the exterior of either, or both, of the native HCV core and its precursor polyprotein.