A replication defective recombinant Ad5 vaccine expressing Ebola virus GP is safe and immunogenic in healthy adults.

Ebola virus causes irregular outbreaks of severe hemorrhagic fever in equatorial Africa. Case mortality remains high; there is no effective treatment and outbreaks are sporadic and unpredictable. Studies of Ebola virus vaccine platforms in non-human primates have established that the induction of protective immunity is possible and safety and human immunogenicity has been demonstrated in a previous Phase I clinical trial of a 1st generation Ebola DNA vaccine. We now report the safety and immunogenicity of a recombinant adenovirus serotype 5 (rAd5) vaccine encoding the envelope glycoprotein (GP) from the Zaire and Sudan Ebola virus species, in a randomized, placebo-controlled, double-blinded, dose escalation, Phase I human study. Thirty-one healthy adults received vaccine at 2×10(9) (n=12), or 2×10(10) (n=11) viral particles or placebo (n=8) as an intramuscular injection. Antibody responses were assessed by ELISA and neutralizing assays; and T cell responses were assessed by ELISpot and intracellular cytokine staining assays. This recombinant Ebola virus vaccine was safe and subjects developed antigen specific humoral and cellular immune responses.

A highly sensitive and versatile virus titration assay in the 96-well microplate format.

This report describes a fast, reproducible, inexpensive and convenient assay system for virus titration in the 96-well format. The micromethod substantially increases assay throughput and improves the data reproducibility. A highly simplified variant of virus quantification is based on immunohistochemical detection of virus amplification foci obtained without use of agarose or semisolid overlays. It can be incorporated into several types of routine virological assays successfully replacing the laborious and time-consuming conventional methods based on plaque formation under semisolid overlays. The method does not depend on the development of CPE and can be accommodated to assay viruses with substantial differences in growth properties. The use of enhanced immunohistochemical detection enabled a five- to six-fold reduction of the total assay time. The micromethod was specifically developed to take advantage of multichannel pipettor use to simplify handling of a large number of samples. The method performs well with an inexpensive low-power binocular, thus offering a routine assay system usable outside of specialized laboratory setting, such as for testing of clinical or field samples. When used in focus reduction-neutralization tests (FRNT), the method accommodates very small volumes of immune serum, which is often a decisive factor in experiments involving small rodent models.

Phase I trial of a melanoma vaccine with gp100(280-288) peptide and tetanus helper peptide in adjuvant: immunologic and clinical outcomes.

A melanoma vaccine composed of HLA-A2-restricted peptide YLEPGPVTA (gp100(280)), with or without a modified T-helper epitope from tetanus toxoid AQYIKANSKFIGITEL, has been evaluated in a Phase I trial to assess safety and immunological response. The vaccines were administered s.c. in either of two adjuvants, Montanide ISA-51 or QS-21, to 22 patients with high-risk resected melanoma (stage IIB-IV). Local and systemic toxicities were mild and transient. We detected CTL responses to the gp100(280) peptide in peripheral blood in 14% of patients. Helper T-cell responses to the tetanus helper peptide were detected in 79% of patients and had a Th1 cytokine profile. One patient with a CTL response to gp100 had a recurrence in a lymph node 2 years later; her nodes contained CD8+ cells reactive to gp100(280) (0.24%), which proliferated in response to peptide. The overall survival of patients is 75% (95% confidence interval, 57-94%) at 4.7 years follow-up, which compares favorably with expected survival. Four of 14 patients who completed at least six vaccines subsequently developed metastases, all of which were solitary and surgically resectable. They remain alive and clinically free of disease at last follow-up. Data from this trial demonstrate immunogenicity of the gp100(280) peptide and suggest that immune responses may persist long-term in some patients. The frequency and magnitude of the CTL response may be improved with more aggressive vaccination regimens. Although this Phase I study was not intended to evaluate clinical benefit, the excellent survival of patients on this protocol suggests the possibility of a benefit that should be assessed in future studies.

Evaluation of peptide vaccine immunogenicity in draining lymph nodes and peripheral blood of melanoma patients.

Many peptide epitopes for cytotoxic T lymphocytes (CTLs) have been identified from melanocytic differentiation proteins. Vaccine trials with these peptides have been limited mostly to those associated with HLA-A2, and immune responses have been detected inconsistently. Cases of clinical regression have been observed after peptide vaccination in some trials, but melanoma regressions have not correlated well with T-cell responses measured in peripheral blood lymphocytes (PBLs). We vaccinated stage IV melanoma patients with a mixture of gp100 and tyrosinase peptides restricted by HLA-A1 (DAEKSDICTDEY), HLA-A2 (YLEPGPVTA and YMDGTMSQV) and HLA-A3 (ALLAVGATK) in an emulsion with GM-CSF and Montanide ISA-51 adjuvant. CTL responses were assessed in PBLs and in a lymph node draining a vaccine site (sentinel immunized node, SIN). We found CTL responses to vaccinating peptides in the SIN in 5/5 patients (100%). Equivalent assays detected peptide-reactive CTLs in PBLs of 2 of these 5 patients (40%). CTLs expanded from the SIN lysed melanoma cells naturally expressing tyrosinase or gp100. We demonstrated immunogenicity for peptides restricted by HLA-A1 and -A3 and for 1 HLA-A2 restricted peptide, YMDGTMSQV. Immune monitoring of clinical trials by evaluation of PBLs alone may under-estimate immunogenicity; evaluation of SIN provides a new and sensitive approach for defining responses to tumor vaccines and correlating these responses with clinical outcomes. This combination of an immunogenic vaccine strategy with a sensitive analysis of CTL responses demonstrates the potential for inducing and detecting anti-tumor immune responses in the majority of melanoma patients.

Analysis of a natural immune response against tumor antigens in a melanoma survivor: lessons applicable to clinical trial evaluations.

The long-term survival of some patients with metastatic melanoma may be attributable in part to cellular immune responses to melanoma antigens. However, little is known about the level of CTL reactivity in vivo that is required for immunological control of tumor progression. In the present report, T-cell responses were evaluated with lymphocytes obtained from tumor-involved nodes and peripheral blood of a long-term melanoma survivor. Using an ELISPOT assay, naturally occurring functional T cells, which recognize the peptide ALLAVGATK (gp100(17-25)) plus two other HLA-A3 restricted peptides, were detected in a tumor-involved lymph node. The ALLAVGATK-reactive T cells were also evaluated by MHC-tetramers staining and were found to be CD8+ CD45RO+ L-selectin(-) CD11a+, suggesting that they are antigen experienced and have a memory phenotype. Unstimulated peripheral blood lymphocytes from the same patient demonstrated no detectable T-cell responses; however, a single stimulation with ALLAVGATK peptide in vitro resulted in a dramatic expansion of peptide-reactive CTLs. This patient, with evidence of tumor-reactive CTLs targeted to several tumor antigens in a tumor-involved lymph node and with evidence of a circulating memory T-cell response, has remained disease-free for 6 years, despite prior bulky nodal metastasis. In contrast, three HLA-A3+ patients with rapidly progressive metastatic melanoma had no detectable T-cell response in tumor-involved nodes or peripheral blood lymphocytes, even after peptide stimulation ex vivo. The presented data are consistent with a systemic polyvalent immune response against tumor in this long-term survivor. These data provide an estimate of the level of CTL response that may be associated with protection from tumor recurrence.

Terminal modifications inhibit proteolytic degradation of an immunogenic MART-1(27-35) peptide: implications for peptide vaccines.

Peptide epitopes for tumor-reactive cytotoxic T-lymphocytes (CTL) have been identified on human cancers and are being used in tumor vaccine trials. However, the pharmacokinetics and pharmacodynamics of such peptides have been inadequately studied. It is predicted that immunogenic tumor peptides would have short half-lives in vivo. The goal of the present work was to evaluate the stability of the immunogenic peptide MART-1(27-35) in fresh normal human plasma (NHP) and to identify modifications that convey protection against enzymatic destruction without loss of immunogenicity. We evaluated the stability of the MART-1(27-35) peptide (AAGIGILTV) and modified forms of that peptide for stability and immune recognition in an in vitro model. The peptides were incubated in plasma for varied time intervals and evaluated for their ability to reconstitute the epitope for MART-1(27-35)-reactive CTL. Loss of CTL reactivity signaled loss of immunoreactive peptide. When 1 microM MART-1(27-35) peptide was incubated in plasma prior to pulsing on target cells, CTL reactivity was lost within 3 hr, and the calculated half-life of this peptide was 22 sec. This degradation was mediated by peptidases. The stability of MART-1(27-35) was markedly prolonged by C-terminal amidation and/or N-terminal acetylation (peptide capping), or by polyethylene-glycol modification (PEGylation) of the C-terminus. These modified peptides were recognized by CTL. The MART-1(27-35) peptide is very unstable in plasma. It is probable that it and other immunogenic peptides will be similarly unstable in vivo. Immunogenicity of these peptides might be enhanced by creating modifications that enhance stability.

Human immunodeficiency virus type 2 glycoprotein enhancement of particle budding: role of the cytoplasmic domain.

Previous studies have shown that the glycoprotein cytoplasmic domains of human immunodeficiency virus type 2 (HIV-2) or simian immunodeficiency virus of macaques modulate biological activities of the viral glycoprotein complex, including syncytium formation, exterior glycoprotein conformation, and glycoprotein incorporation into budding virus particles. We have now utilized a recombinant expression system to study interactions of full-length or truncated HIV-2 glycoproteins with coexpressed HIV-2 Gag proteins which self-assemble and bud as virus-like particles. Interestingly, budding of HIV-2 virus-like particles from cells was enhanced 5- to 24-fold when Gag was coexpressed with the full-length HIV-2 glycoprotein, compared with Gag expressed either alone or with a truncated HIV-2 glycoprotein. The results obtained in this model system indicate that an additional effect of the lengthy cytoplasmic domain of the glycoprotein of HIV-2 is enhancement of particle budding. We speculate that the cytoplasmic domain of the viral glycoprotein of HIV-2 enhances budding by (i) potentiation of Gag structure or function or (ii) membrane modulation.

Assembly of SIV virus-like particles containing envelope proteins using a baculovirus expression system.

The requirements for SIV particle assembly and envelope incorporation were investigated using a baculovirus expression system. The Pr56gag precursor protein expressed under control of the polyhedrin promoter (pPolh) produced high levels of immature retrovirus-like particles (VLP) upon expression in Sf9 insect cells. To determine the optimal conditions for envelope protein (Env) incorporation into VLP, two recombinant baculoviruses expressing the SIV envelope protein under control of a very late pPolh or a hybrid late/very late capsid/polyhedrin (Pcap/polh) promoter and a recombinant expressing a truncated form of the SIV envelope protein (Envt) under the hybrid Pcap/polh promoter were compared. We have observed that utilization of the earlier hybrid promoter resulted in higher levels of Env expression on the cell surface and its incorporation into budding virus particles. We have also found that the Envt protein is transported to the cell surface of insect cells and incorporated into VLP more efficiently than full-length Env. In addition, we examined the effect of coexpression of the protease furin, which has been implicated in the proteolytic cleavage of the Env precursor gp160 in mammalian cells. Coexpression of furin in insect cells resulted in more efficient proteolytic cleavage into gp120 and gp41, and the cleaved proteins were incorporated into VLP.

Cytoplasmic domain truncation enhances fusion activity by the exterior glycoprotein complex of human immunodeficiency virus type 2 in selected cell types.

To investigate the glycoprotein determinants of viral cytopathology, we constructed chimeric env genes between a noncytopathic strain of human immunodeficiency virus type 2 (HIV-2), designated HIV-2/ST, and a highly fusogenic and cytopathic variant derived from this virus. Expression of the resulting chimeric glycoproteins indicated that efficient syncytium formation in the human T-cell line Sup T1 mapped to the C-terminal region of the transmembrane (TM) glycoprotein subunit. In this region, the wild-type and cytopathic ST glycoproteins differed by only four amino acids and by the presence of a premature termination codon in the cytopathic variant. Subsequent site-directed mutagenesis indicated that the cytoplasmic domain truncation was responsible for the enhanced fusion activity. This modification, however, increased the fusion activity of the glycoprotein only in Sup T1 cells (in which the ST variant arose) but not in Molt 4 clone 8 or peripheral blood mononuclear cells. These observations indicate that the length of the cytoplasmic domain of the HIV-2 glycoprotein modulates the fusion activity of the exterior glycoprotein complex in a cell-specific manner. Such adaptability appears to permit the emergence of fusogenic variants during HIV-2 passage in vitro and may also regulate viral growth or cytopathic effects in selected cell types during natural infection in vivo.