Characterization of Vero cell growth and death in bioreactor with serum-containing and serum-free media.

The density of viable cells in a culture results from a balance between cell proliferation and cell death. The aim of this study was to characterize and compare these two phenomena in Vero cell cultures in one serum containing medium (ScA) and one serum free medium (SfB) in bioreactors. Cell growth was evaluated by cell counting(after crystal violet staining) and cell cycle analysis.

Kinetics and metabolic specificities of Vero cells in bioreactor cultures with serum-free medium.

The aim of this study was to understand the metabolism kinetics of Vero cells grown on microcarriers in bioreactors in serum-free medium (SFM). We sought to determine what nutrients are essential for Vero cells and how they are consumed. Contrary to glucose and to most of the amino acids, glutamine and serine were very quickly depleted in this medium and can be supposed to be responsible for cell apoptosis.

Safety, immunogenicity and protective efficacy in mice of a new cell-cultured Lister smallpox vaccine candidate.

It is now difficult to manufacture the first-generation smallpox vaccine, as the process could not comply with current safety and manufacturing regulations. In this study, a candidate non-clonal second-generation smallpox vaccine developed by Sanofi-Pasteur from the Lister strain has been assessed using a cowpox virus challenge in mice. We have observed similar safety, immunogenicity and protection (from disease and death) after a short or long interval following vaccination, as well as similar virus clearance post-challenge, with the second-generation smallpox vaccine candidate as compared to the traditional vaccine used as a benchmark.

Safety and immunogenicity of a live recombinant canarypox virus expressing HIV type 1 gp120 MN MN tm/gag/protease LAI (ALVAC-HIV, vCP205) followed by a p24E-V3 MN synthetic peptide (CLTB-36) administered in healthy volunteers at low risk for HIV infection. AGIS Group and L'Agence Nationale de Recherches sur Le Sida.

A live recombinant canarypox vector expressing HIV-1 gpl20 MN tm/gag/protease LAI (ALVAC-HIV, vCP205) alone or boosted by a p24E-V3 MN synthetic peptide (CLTB-36) was tested in healthy volunteers at low risk for HIV infection for their safety and immunogenicity. Both antigens were well tolerated. ALVAC-HIV (vCP205) induced low levels of neutralizing antibodies against HIV-1 MN in 33% of the volunteers.

Fine specificity of anti-V3 antibodies induced in chimpanzees by HIV candidate vaccines.

The fine specificity of the anti-V3 antibody responses induced in chimpanzees immunized by various human immunodeficiency type 1 (HIV-1) candidate vaccines and challenged by heterologous strains of HIV-1 was analyzed by enzyme-linked immunosorbent assay (ELISA) and Pepscan epitope mapping. Two chimpanzees immunized with the recombinant canarypox virus ALVAC-HIV (vCP125) expressing gp160MN and boosted with purified gp160MN/LAI alone, then with both immunogens in combination, were not protected against challenge with HIV-1 SF2. Their sera mainly recognized one epitope of the V3 loop, located in the NH2-terminal half.

Restricted specificity of anti-V3 antibodies induced in humans by HIV candidate vaccines.

We analyzed the fine specificity of anti-V3 antibodies elicited in three different species (human, guinea pig, and macaque) by various HIV candidate vaccines. Following immunization with recombinant canarypox virus expressing gp160MN or with recombinant gp160MN/LAI, this antibody response was shown to be directed against the NH2-terminal region of the V3 loop. Although this response was increased by a prime-boost regimen using immunization with canarypox expressing gp160 followed by an rgp160 boost, its specificity remained restricted mainly to the recognition of this region of the V3 loop.

Challenge of chimpanzees immunized with a recombinant canarypox-HIV-1 virus.

To evaluate the potential protective efficacy of a live recombinant human immunodeficiency virus type 1 (HIV-1) canarypox vaccine candidate, two chimpanzees were immunized five times with ALVAC-HIV-1 vCP250, a recombinant canarypox virus that expresses the HIV-1[IIIB(LAI)] gp120/TM, gag, and protease gene products. One month after the last booster inoculation, the animals were challenged by intravenous injection of cell-associated virus in the form of peripheral blood mononuclear cells from an HIV-1[IIIB(LAI)]-infected chimpanzee. One chimpanzee with a neutralizing antibody titer to HIV-1[IIIB(LAI)] of 128 at the time of challenge was protected, whereas both the second animal, with a neutralizing antibody titer of 32, and a naive control animal became infected.

Failure of a human immunodeficiency virus type 1 (HIV-1) subtype B-derived vaccine to prevent infection of chimpanzees by an HIV-1 subtype E strain.

Generation of an effective vaccine against human immunodeficiency virus type 1 (HIV-1) must overcome problems associated with extensive genetic diversity. Although we previously reported vaccine-induced protection of chimpanzees against infection with an HIV-1 strain different from the one used to make the immunogens, both the HIV-1 vaccine and challenge strains were classified within subtype B. To determine whether the HIV-1-specific immunity elicited might also prevent infection by a strain of HIV-1 from a different clade, the same chimpanzees were given booster inoculations with the rgp160-MN/LAI (recombinant hybrid gp160 molecule) and V3-MN immunogens and then were challenged by intravenous inoculation of a comparable dose of a subtype E HIV-1 from the Central African Republic.

African green monkey kidney (Vero) cells provide an alternative host cell system for influenza A and B viruses.

The preparation of live, attenuated human influenza virus vaccines and of large quantities of inactivated vaccines after the emergence or reemergence of a pandemic influenza virus will require an alternative host cell system, because embryonated chicken eggs will likely be insufficient and suboptimal. Preliminary studies indicated that an African green monkey kidney cell line (Vero) is a suitable system for the primary isolation and cultivation of influenza A viruses (E. A.

Human safety and immunogenicity of a canarypox-rabies glycoprotein recombinant vaccine: an alternative poxvirus vector system.

Avian poxvirus recombinants undergo abortive replication in nonavian cells, yet can achieve expression of extrinsic gene products. Canarypox-vectored vaccines have been innocuous and immunogenic in several mammalian species. ALVAC-RG, a canarypox recombinant expressing the rabies glycoprotein gene, was inoculated intramuscularly into adult volunteers on days 0, 28, and 180.

Safety and immunogenicity of a recombinant HIV type 1 glycoprotein 160 boosted by a V3 synthetic peptide in HIV-negative volunteers.

The safety and the immunogenicity of a recombinant hybrid envelope glycoprotein MN/LAI (rgp160) followed by booster injections of a V3 (MN) linear peptide were evaluated in HIV-negative adults at low risk for HIV infection. Volunteers received either rgp160 in alum at 0, 1, and 6 months (group A), rgp160 at 0 and 1 months followed by V3 at 3 and 6 months formulated in alum (group B), or in Freund's incomplete adjuvant (FIA) (group C). Local and systemic reactions were mild to moderate and resolved within the first 72 hr after immunization.

Vaccine-induced protection of chimpanzees against infection by a heterologous human immunodeficiency virus type 1.

The extraordinary genetic diversity of human immunodeficiency virus type 1 (HIV-1) is a major problem to overcome in the development of an effective vaccine. In the most reliable animal model of HIV-1 infection, chimpanzees were immunized with various combinations of HIV-1 antigens, which were derived primarily from the surface glycoprotein, gp160, of HIV-1 strains LAI and MN. The immunogens also included a live recombinant canarypox virus expressing a gp160-MN protein.

Replication of influenza A viruses in a green monkey kidney continuous cell line (Vero).

A Vero cell line was investigated as a suitable host system for primary isolation and cultivation of influenza A viruses. The efficiency of primary isolation for currently circulating (H3N2) strains was similar in Vero and MDCK cells. Of 72 egg-adapted strains investigated, 90.

Biological and immunogenic properties of a canarypox-rabies recombinant, ALVAC-RG (vCP65) in non-avian species.

A canarypox-based (ALVAC) recombinant expressing the rabies G glycoprotein has been utilized to assess in vitro and in vivo biological properties of the canarypox virus vector system. In vitro studies have shown that no replication of the virus can be detected on six human-derived cell lines, nor can the virus be readily adapted to replicate on non-avian cells. Expression of the rabies G can be detected on all cell lines analyzed in the absence of productive viral replication.

A prime-boost approach to HIV preventive vaccine using a recombinant canarypox virus expressing glycoprotein 160 (MN) followed by a recombinant glycoprotein 160 (MN/LAI). The AGIS Group, and l'Agence Nationale de Recherche sur le SIDA.

The safety and the immunogenicity of a recombinant canarypox live vector expressing the human immunodeficiency virus type 1 (HIV-1) gp160 gene from the MN isolate, ALVAC-HIV (vCP125), followed by booster injections of a soluble recombinant hybrid envelope glycoprotein MN/LAI (rgp160), were evaluated in vaccinia-immune, healthy adults at low risk for acquiring HIV-1 infection. Volunteers (n = 20) received vCP125 (10(6) TCID50) at 0 and 1 month, followed randomly by rgp160 formulated in alum or in Freund's incomplete adjuvant (FIA) at 3 and 6 months. Local and systemic reactions were mild or moderate and resolved within the first 72 hr after immunization.

The safety and use of canarypox vectored vaccines.

ALVAC recombinants have been administered to humans and animals by parenteral and oral routes without giving signs of replication, systemic dissemination or severe reaction. In principle, it should be impossible for canarypox recombinants to disseminate in the environment as they would not be synthesised in mammalian cells as complete virus. Canarypox vectors have been safe for humans, in whom there has been no evidence of replication, but more work needs to be done to prove absence of replication.

Highly attenuated poxvirus vectors: NYVAC, ALVAC and TROVAC.

Three highly attenuated and efficacious poxvirus-based vectors, NYVAC, ALVAC and TROVAC, are available for targeted applications as recombinant vaccines in both human and veterinary medicine. The attenuated phenotype of the three vectors is consistent with safe use for vaccination purposes, for the vaccinee, for unvaccinated contacts, and for introduction into the environment. The precise deletion of virulence and host range genes in the NYVAC vector precludes reversion to the virulent phenotype by back mutation.

Early region 3-replacement adenovirus recombinants are less pathogenic in cotton rats and mice than early region 3-deleted viruses.

Background: Adenovirus type-5 (Ad5) recombinant viruses with replacement of the 1.9 kb XbaI fragment in the early region 3 (E3) by foreign genes have been constructed with the ultimate goal of inducing immune responses to the product of the inserted gene against a variety of virus infections. The pathogenicity of these recombinants, however, has not been studied.

Immunisation with canarypox virus expressing rabies glycoprotein.

Poxviruses have many useful features as vectors for genes that carry immunising antigens from other viruses, such as ease of production and induction of cellular and humoral immunity, but there is concern about the safety of vaccinia virus. We turned to an avian poxvirus (canarypox); this virus undergoes abortive replication in mammalian cells that enables presentation of early gene products to the immune system. Canarypox virus was used as a vector for the rabies glycoprotein G gene.

NYVAC: a highly attenuated strain of vaccinia virus.

A highly attenuated vaccinia virus strain, NYVAC (vP866), was derived from a plaque-cloned isolate of the Copenhagen vaccine strain by the precise deletion of 18 open reading frames (ORFs) from the viral genome. Among the ORFs deleted from NYVAC (vP866) are two genes involved in nucleotide metabolism, the thymidine kinase (ORF J2R) and the large subunit of the ribonucleotide reductase (ORF I4L); the gene encoding the viral hemagglutinin (ORF A56R); the remnant (ORF A26L) of a highly expressed gene responsible for the formation of A-type inclusion bodies; the disrupted gene (ORFs B13R/B14R) normally encoding a serine protease inhibitor; and a block of 12 ORFs bounded by two known viral host range regulatory functions (ORFs C7L through K1L). Within this block a secretory protein (ORF N1L) implicated in viral virulence and a functional complement 4b binding protein (ORF C3L) are encoded.

Comparison of the ability of formalin-inactivated respiratory syncytial virus, immunopurified F, G and N proteins and cell lysate to enhance pulmonary changes in Balb/c mice.

Formalin-inactivated respiratory syncytial virus (FI-RSV), a lysate of HEp-2 cells and proteins F, G and N, immunopurified from infected cell cultures, were compared for their ability to prevent infection and to enhance changes in lung cytology associated with RSV challenge. Mice were immunized at three weekly intervals with serial dilutions of the preparations and challenged by the nasal route 1 week after the last injection; their lungs were analysed 4 days later. The concentration of the immunogens was adjusted to test at least a range of 2 to 500 ng of proteins per injection.