Production, quality control, stability and pharmacotoxicity of cGMP-produced Plasmodium falciparum AMA1 FVO strain ectodomain expressed in Pichia pastoris.

Plasmodium falciparum apical membrane antigen 1 (PfAMA1) is a leading asexual blood stage vaccine candidate for malaria. In preparation for clinical trials, PfAMA1 ectodomain (amino acid 25-545, FVO strain) was produced in Pichia pastoris by 35L scale fed batch fermentation under current Good Manufacturing Practice (cGMP). Fermentation was followed by a three-step chromatographic purification procedure resulting in a yield of 5.8g of purified protein. As judged by size exclusion chromatography, the cGMP-product comprised >95% PfAMA1 monomer, the remainder being predominantly PfAMA1 dimer. In SDS-PAGE two main bands of 68 and 70kDa and some minor bands were evident. Under reducing conditions a site of limited proteolytic cleavage within a disulphide bonded region became evident; less than 15% of the protein had this internal cleavage. By mass-spectrometric analysis, all bands analyzed in overloaded SDS-PAGE gels comprised PfAMA1 derived products. The protein was quantitatively bound by immobilized 4G2, a monoclonal antibody reactive with a reduction sensitive conformational determinant. The lyophilized product was stable for over 1 year. Immunopotency did not diminish, and storage did not lead to alterations in the behaviour of the protein upon formulation with adjuvants selected for Phase I clinical evaluation. These formulations also showed no pharmacotoxicity in rabbits. The final product conformed to preset criteria and was judged suitable for use in human clinical trials.

Phase I/II studies to evaluate safety and immunogenicity of a recombinant gp350 Epstein-Barr virus vaccine in healthy adults.

Two double-blind randomised controlled studies (phase I and I/II) were performed to assess for the first time the safety and immunogenicity of a recombinant subunit gp350 Epstein-Barr virus (EBV) vaccine in 148 healthy adult volunteers. All candidate vaccine formulations had a good safety profile and were well tolerated, with the incidence of solicited and unsolicited symptoms within a clinically acceptable range. One serious adverse event was reported in the phase I trial which was considered to be of suspected relationship to vaccination. The gp350 vaccine formulations were immunogenic and induced gp350-specific antibody responses (including neutralising antibodies).

Recombinant gp350 vaccine for infectious mononucleosis: a phase 2, randomized, double-blind, placebo-controlled trial to evaluate the safety, immunogenicity, and efficacy of an Epstein-Barr virus vaccine in healthy young adults.

BACKGROUND: To date, there is no commercially available vaccine to prevent infectious mononucleosis, a disease frequently induced by Epstein-Barr virus (EBV) infection in adolescents or adults devoid of preexisting immunity to the virus. METHODS: A total of 181 EBV-seronegative, healthy, young adult volunteers were randomized in a double-blind fashion to receive either placebo or a recombinant EBV subunit glycoprotein 350 (gp350)/aluminum hydroxide and 3-O-desacyl-4'-monophosphoryl lipid A (AS04) candidate vaccine in a 3-dose regimen. RESULTS: The vaccine had demonstrable efficacy (mean efficacy rate, 78.0% [95% confidence interval {CI}, 1.0%-96.0%]) in preventing the development of infectious mononucleosis induced by EBV infection, but it had no efficacy in preventing asymptomatic EBV infection. One month after receipt of the final dose of gp350 vaccine, 98.7% of subjects showed seroconversion to anti-gp350 antibodies (95% CI, 85.5%-97.9%), and they remained anti-gp350 antibody positive for >18 months. Furthermore, there were no concerns regarding the safety or reactogenicity of the gp350/AS04 vaccine. CONCLUSION: These data support the clinical feasibility of using an EBV vaccine to prevent infectious mononucleosis. TRIAL REGISTRATION: ClinicalTrials.gov identifier: NCT00430534.

Immunogenicity of a recombinant varicella-zoster virus gE-IE63 fusion protein, a putative vaccine candidate against primary infection and zoster reactivation.

The varicella-zoster virus (VZV) envelope glycoprotein E (gE) and immediate early protein 63 (IE63) are well known targets for specific humoral and cell-mediated immune responses during VZV infection and latency, respectively. The present study evaluated the immunogenicity of an engineered chimeric recombinant gE-IE63 (recgE-IE63) protein secreted from CHO cells, wherein a soluble form of gE, deleted of its anchor and cytoplasmic domains was fused to IE63. Guinea pig vaccinations with adjuvanted recgE-IE63 elicited a strong and specific humoral immune response directed to each counterpart. Sera from recgE-IE63-immunized animals neutralized cell-free VZV. This neutralizing capacity was dependent only on the recgE moiety as serum depletions on recgE-immobilized sepharose totally abolished VZV neutralization. The cell-mediated immune response induced by recgE-IE63 was evaluated in lymphoproliferation assays. An antigen-specific proliferative response was demonstrated after lymphocyte stimulation with recIE63 but not with recgE. We conclude that recombinant chimeric recgE-IE63 induced both humoral and cell-mediated immune responses and thus could constitute a putative subunit vaccine candidate against VZV primary infection and zoster reactivation.