Cytotoxic T-cell response to simian immunodeficiency virus by cynomolgus macaque monkeys immunized with recombinant vaccinia virus.

Simian immunodeficiency virus (SIV) gag-specific major histocompatibility complex (MHC)-restricted cytotoxic T-lymphocyte (CTL) activity was elicited in four out of six cynomolgus macaques after two immunizations with SIV gag recombinant vaccinia virus (rVV). No activity could be seen in three out of three non-immunized control animals. Low levels of anti-gag antibody were also seen in the same four responding animals. Virus-specific, MHC-restricted CTL are thought to give some protection and to assist in recovery in viral infection, and the induction of such CTL following vaccination with a single viral protein should act as an encouragement to those proposing similar vaccination studies in man.

An HIV-1 and HIV-2 cross-reactive cytotoxic T-cell epitope.

The HLA-B27-restricted HIV gag cytotoxic T-lymphocyte (CTL) epitope, 265-279, is highly conserved amongst HIV-1 isolates, only one, HIV-1ELI, having a single amino acid substitution. Over the same region HIV-2 differs by five amino acids. As a broadly cross-protective AIDS vaccine should protect against infection from all isolates of HIV-1 and HIV-2, we tested CTL specific for the HIV-1 265-279 epitope with peptide analogues from HIV-1ELI, HIV-2 and two simian immunodeficiency virus (SIV) isolates, and with recombinant vaccinia viruses expressing the respective gag genes, to determine whether there was any cross-reactivity for this CTL epitope. CTL from the HIV-1-infected donor could recognize the HIV-1ELI peptide, the HIV-2 peptide and recombinant vaccinia virus-infected target and one of the two SIV peptide-treated targets. Epitopes that exhibit such cross-reactivity may be valuable in vaccine design.

Phase I safety and immunogenicity trial of FMP1/AS02A, a Plasmodium falciparum MSP-1 asexual blood stage vaccine.

We report the first safety and immunogenicity trial of the Plasmodium falciparum malaria blood stage vaccine candidate, FMP1/AS02A consisting of the FMP1 antigen, an Escherichia coli-expressed His-tagged fusion protein from the 42 kDa C-terminal fragment from the 3D7 clone of the merozoite surface protein 1 formulated in the AS02A adjuvant. An open label, prospective, single-center Phase I dose escalation trial of FMP1/AS02A was conducted in 15 adult malaria-naïve human volunteers to assess safety, reactogenicity, and immunogenicity. The vaccine was safe and well-tolerated and no serious adverse events were observed. The vaccine induced high-titer ELISA and IFA responses in all volunteers. Proliferative and ELISPOT responses were induced to vaccine antigen. Biologically active antibodies were induced as measured by GIA. This study establishes the foundation to further evaluate and measure the vaccine's ability to reduce morbidity and mortality in target populations directly affected by P. falciparum malaria.

Cytotoxic T cell immunity to human cytomegalovirus glycoprotein B.

Human cytomegalovirus (HCMV) is associated with significant morbidity and mortality following immunosuppression and in pregnancy. HCMV infection may be accompanied by acute disease but persists asymptomatically. Cytotoxic T lymphocytes (CTL) appear to be an important immune effector mechanism in maintaining the normal host-virus equilibrium. Glycoprotein B may be an important target for future subunit vaccines as it has been found to elicit both neutralising antibody and CTL responses. We therefore studied the ability of normal asymptomatic HCMV-seropositive individuals and women throughout pregnancy to determine the presence of HCMV and gB-specific CTL responses. CTL effector cells were induced by stimulation of peripheral blood mononuclear cells (PBMC) with AD169 HCMV-infected cells and gB-specific CTL were identified using chromium labeled, vac.gB-infected cells. In 7 HCMV-seropositive individuals, HCMV-specific CTL were identified. Three of the 7 individuals which lysed HCMV-infected cells lysed vac.gB-infected B cells. However, vac.gB-infected autologous fibroblasts, which only present MHC class I, were not killed. Using MHC class I single allele targets, no specific lytic response was observed, suggesting a MHC class II restricted CTL response. Flow cytometric analysis showed the gB-specific effector cell phenotype to be CD3+, CD4+, CD8-. In conclusion, a gB-specific CTL lytic response was identified in seropositive individuals which in most cases was MHC class II-restricted.

The GAG precursor of simian immunodeficiency virus assembles into virus-like particles.

To examine the potential role of the GAG precursor polyprotein in morphogenesis and assembly of the simian immunodeficiency virus (SIV), we have expressed the gag gene of SIVMac using a baculovirus expression vector. Infection of insect cells with recombinant virus containing the entire gag gene results in high expression of the GAG precursor protein, Pr57gag. The recombinant protein is myristylated and is released in the culture supernatant in an insoluble particulate form. A point mutation in the N-terminal glycine codon (Gly----Ala) inhibits myristylation. This mutated product is highly expressed but is not found in the culture supernatant. Electron microscopy and immunogold labelling of infected cells show that the native Pr57gag protein assembles into 100-120 nm virus-like particles that bud from the cell plasma membrane and are released in the culture supernatant. The unmyristylated protein also assembles into particulate structures which only accumulate inside the cells. These results demonstrate that the unprocessed GAG precursor of SIV can spontaneously assemble into particles in the absence of other viral proteins. Myristylation of the Pr57gag precursor is necessary for its association with the cell plasma membrane, for budding and for extracellular release.

Efficacy of recombinant circumsporozoite protein vaccine regimens against experimental Plasmodium falciparum malaria.

After initial successful evaluation of the circumsporozoite-based vaccine RTS,S/SBAS2, developed by SmithKline Beecham Biologicals with the Walter Reed Army Institute of Research, protective efficacy of several regimens against Plasmodium falciparum challenge was determined. A controlled phase 1/2a study evaluated 1 or 2 standard doses of RTS,S/SBAS2 in 2 groups whose members received open-label therapy and 3 immunizations in blinded groups who received standard, one-half, or one-fifth doses. RTS,S/SBAS2 was safe and immunogenic in all groups. Of the 41 vaccinees and 23 control subjects who underwent sporozoite challenge, malaria developed in 7 of 10 who received 1 dose, in 7 of 14 who received 2 doses, in 3 of 6 who received 3 standard doses, in 3 of 7 who received 3 one-half doses, in 3 of 4 who received 3 one-fifth doses, and in 22 of 23 control subjects. Overall protective efficacy of RTS,S/SBAS2 was 41% (95% confidence interval, 22%-56%; P=.0006). This and previous studies have shown that 2 or 3 doses of RTS,S/SBAS2 protect against challenge with P. falciparum sporozoites.

Potent induction of focused Th1-type cellular and humoral immune responses by RTS,S/SBAS2, a recombinant Plasmodium falciparum malaria vaccine.

The RTS,S/SBAS2 vaccine confers sterile protection against Plasmodium falciparum sporozoite challenge. The mechanisms underlying this are of great interest, yet little is known about the immune effector mechanisms induced by this vaccine. The immune responses induced by RTS,S/SBAS2 were characterized in 10 malaria-naive volunteers. Several epitopes in the circumsporozoite protein (CSP) were identified as targets of cultured interferon (IFN)-gamma-secreting CD4+ T cells. RTS,S-specific IFN-gamma-secreting effector T cells were induced in 8 subjects; this ex vivo response mapped to a single peptide in Th2R. CSP-specific CD8+ cytotoxic T lymphocytes were not detected. RTS, S-specific IFN-gamma production was universal, whereas interleukin-4 and -5 production was rare. RTS,S-specific lymphoproliferative responses and antibodies to CSP were strongly induced in all volunteers. Responses waned with time but were boostable. Thus, RTS, S/SBAS2 is a potent inducer of Th1-type cellular and humoral immunity. These results highlight possible immune mechanisms of protection and have important implications for vaccine design in general.

Efficacy of RTS,S/AS02 malaria vaccine against Plasmodium falciparum infection in semi-immune adult men in The Gambia: a randomised trial.

BACKGROUND: RTS,S/AS02 is a pre-erythrocytic malaria vaccine based on the circumsporozoite surface protein of Plasmodium falciparum fused to HBsAg, incorporating a new adjuvant (AS02). We did a randomised trial of the efficacy of RTS,S/AS02 against natural P. falciparum infection in semi-immune adult men in The Gambia. METHODS: 306 men aged 18-45 years were randomly assigned three doses of either RTS,S/AS02 or rabies vaccine (control). Volunteers were given sulfadoxine/pyrimethamine 2 weeks before dose 3, and kept under surveillance throughout the malaria transmission season. Blood smears were collected once a week and whenever a volunteer developed symptoms compatible with malaria. The primary endpoint was time to first infection with P. falciparum. Analysis was per protocol. FINDINGS: 250 men (131 in the RTS,S/AS02 group and 119 in the control group) received three doses of vaccine and were followed up for 15 weeks. RTS,S/AS02 was safe and well tolerated. P. falciparum infections occurred significantly earlier in the control group than the RTS,S/AS02 group (Wilcoxon's test p=0.018). Vaccine efficacy, adjusted for confounders, was 34% (95% CI 8.0-53, p=0.014). Protection seemed to wane: estimated efficacy during the first 9 weeks of follow-up was 71% (46-85), but decreased to 0% (-52 to 34) in the last 6 weeks. Vaccination induced strong antibody responses to circumsporozoite protein and strong T-cell responses. Protection was not limited to the NF54 parasite genotype from which the vaccine was derived. 158 men received a fourth dose the next year and were followed up for 9 weeks; during this time, vaccine efficacy was 47% (4-71, p=0.037). INTERPRETATION: RTS,S/AS02 is safe, immunogenic, and is the first pre-erythrocytic vaccine to show significant protection against natural P. falciparum infection.