A whole virus pandemic influenza H1N1 vaccine is highly immunogenic and protective in active immunization and passive protection mouse models.

The recent emergence and rapid spread of a novel swine-derived H1N1 influenza virus has resulted in the first influenza pandemic of this century. Monovalent vaccines have undergone preclinical and clinical development prior to initiation of mass immunization campaigns. We have carried out a series of immunogenicity and protection studies following active immunization of mice, which indicate that a whole virus, nonadjuvanted vaccine is immunogenic at low doses and protects against live virus challenge. The immunogenicity in this model was comparable to that of a whole virus H5N1 vaccine, which had previously been demonstrated to induce high levels of seroprotection in clinical studies. The efficacy of the H1N1 pandemic vaccine in protecting against live virus challenge was also seen to be equivalent to that of the H5N1 vaccine. The protective efficacy of the H1N1 vaccine was also confirmed using a severe combined immunodeficient (SCID) mouse model. It was demonstrated that mouse and guinea pig immune sera elicited following active H1N1 vaccination resulted in 100% protection of SCID mice following passive transfer of immune sera and lethal challenge. The immune responses to a whole virus pandemic H1N1 and a split seasonal H1N1 vaccine were also compared in this study. It was demonstrated that the whole virus vaccine induced a balanced Th-1 and Th-2 response in mice, whereas the split vaccine induced mainly a Th-2 response and only minimal levels of Th-1 responses. These data supported the initiation of clinical studies with the same low doses of whole virus vaccine that had previously been demonstrated to be immunogenic in clinical studies with a whole virus H5N1 vaccine.

Vero cell platform in vaccine production: moving towards cell culture-based viral vaccines.

The development of cell culture systems for virus propagation has led to major advances in virus vaccine development. Primary and diploid cell culture systems are now being replaced by the use of continuous cell lines (CCLs). These substrates are gaining increasing acceptance from regulatory authorities as improved screening technologies remove fears regarding their potential oncogenic properties. The Vero cell line is the most widely accepted CCL by regulatory authorities and has been used for over 30 years for the production of polio and rabies virus vaccines. The recent licensure of a Vero cell-derived live virus vaccine (ACAM2000, smallpox vaccine) has coincided with an explosion in the development of a range of new viral vaccines, ranging from live-attenuated pediatric vaccines against rotavirus infections to inactivated whole-virus vaccines against H5N1 pandemic influenza. These developments have illustrated the value of this cell culture platform in the rapid development of vaccines against a range of virus diseases.

Cell culture (Vero) derived whole virus (H5N1) vaccine based on wild-type virus strain induces cross-protective immune responses.

The rapid spread and the transmission to humans of avian influenza virus (H5N1) have induced world-wide fears of a new pandemic and raised concerns over the ability of standard influenza vaccine production methods to rapidly supply sufficient amounts of an effective vaccine. We report here on a robust and flexible strategy which uses wild-type virus grown in a continuous cell culture (Vero) system to produce an inactivated whole virus vaccine. Candidate vaccines based on clade 1 and clade 2 influenza H5N1 strains were developed and demonstrated to be highly immunogenic in animal models. The vaccines induce cross-neutralising antibodies, highly cross-reactive T-cell responses and are protective in a mouse challenge model not only against the homologous virus but also against other H5N1 strains, including those from another clade. These data indicate that cell culture-grown whole virus vaccines, based on the wild-type virus, allow the rapid high yield production of a candidate pandemic vaccine.

The preclinical testing of a formaldehyde inactivated Ross River virus vaccine designed for use in humans.

Ross River virus was grown in industrial facilities in vaccine-certified Vero cells in the absence of serum, inactivated using standard formalin-inactivation protocols, treated with Benzonase to digest host cell DNA and purified on a sucrose gradient. Mice given two subcutaneous injections of 0.625 microg of this vaccine or two doses of 0.156 microg vaccine with aluminium hydroxide adjuvant failed to develop a detectable viraemia after intravenous challenge with 10(6)TCID50 of the prototype strain of Ross River virus (T48). Guinea pigs immunised with one or two10 microg doses of vaccine with adjuvant also failed to develop a detectable viraemia following a similar challenge. The levels of neutralising antibody (neutralisation index 1.9-3.1) in the mice protected against challenge with 10(6)TCID50 Ross River virus were similar to those in 16 former epidemic polyarthritis patients (1.1-3.5) who had not experienced a second clinical infection with Ross River virus in the 20 years following their initial infection.

A double-inactivated whole virus candidate SARS coronavirus vaccine stimulates neutralising and protective antibody responses.

A double-inactivated, candidate whole virus vaccine against severe acute respiratory syndrome associated coronavirus (SARS-CoV) was developed and manufactured at large scale using fermenter cultures of serum protein free Vero cells. A two step inactivation procedure involving sequential formaldehyde and U.V. inactivation was utilised in order to ensure an extremely high safety margin with respect to residual infectivity. The immunogenicity of this double-inactivated vaccine was characterised in the mouse model. Mice that were immunised twice with the candidate SARS-CoV vaccine developed high antibody titres against the SARS-CoV spike protein and high levels of neutralising antibodies. The use of the adjuvant Al(OH)3 had only a minor effect on the immunogenicity of the vaccine. In addition, cell mediated immunity as measured by interferon-gamma and interleukin-4 stimulation, was elicited by vaccination. Moreover, the vaccine confers protective immunity as demonstrated by prevention of SARS-CoV replication in the respiratory tract of mice after intranasal challenge with SARS-CoV. Protection of mice was correlated to antibody titre against the SARS-CoV S protein and neutralising antibody titre.

ACAM2000 clonal Vero cell culture vaccinia virus (New York City Board of Health strain)--a second-generation smallpox vaccine for biological defense.

The threat of smallpox as a biological weapon has spurred efforts to create stockpiles of vaccine for emergency preparedness. In lieu of preparing vaccine in animal skin (the original method), we cloned vaccinia virus (New York City Board of Health strain, Dryvax by plaque purification and amplified the clone in cell culture. The overarching goal was to produce a modern vaccine that was equivalent to the currently licensed Dryvax in its preclinical and clinical properties, and could thus reliably protect humans against smallpox. A variety of clones were evaluated, and many were unacceptably virulent in animal models. One clonal virus (ACAM1000) was selected and produced at clinical grade in MRC-5 human diploid cells. ACAM1000 was comparable to Dryvax in immunogenicity and protective activity but was less neurovirulent for mice and nonhuman primates. To meet requirements for large quantities of vaccine after the events of September 11th 2001, the ACAM1000 master virus seed was used to prepare vaccine (designated ACAM2000) at large scale in Vero cells under serum-free conditions. The genomes of ACAM1000 and ACAM2000 had identical nucleotide sequences, and the vaccines had comparable biological phenotypes. ACAM1000 and ACAM2000 were evaluated in three Phase 1 clinical trials. The vaccines produced major cutaneous reactions and evoked neutralizing antibody and cell-mediated immune responses in the vast majority of subjects and had a reactogenicity profile similar to that of Dryvax.