Preparation of vaccines against H5N1 influenza.

In response to the pandemic warning provided by the highly pathogenic H5N1 influenza virus infections in Hong Kong, there were world-wide attempts to develop vaccines. Three strategies were followed and although each was associated with some success, there were also some problems. Pre-clinical vaccine efficacy results are presented from one such strategy, that of using an apathogenic H5N3 avian strain for vaccine production.

Clinical safety issues of measles, mumps and rubella vaccines.

The clinical safety of measles and measles-mumps-rubella vaccines has been questioned in recent reports that propose a possible link between measles virus or measles vaccines and the occurrence of juvenile Crohn disease and autism. This article reviews the outcomes of several laboratory investigations which were carried out independently to identify the presence or absence of measles virus in the intestinal tissues derived from cases of inflammatory bowel disease. One research group reported the presence of measles virus particles and genomic RNA in inflammatory bowel disease tissues, but this could not be confirmed by other groups, despite use of techniques that are highly specific and sensitive for the detection of measles virus nucleic acid in clinical specimens down to the molecular level. Based on the published data reviewed here, it can be concluded that there is no direct association between measles virus or measles vaccines and the development of Crohn disease, a conclusion which is supported by most epidemiological findings.

Single radial immunodiffusion as a method for the assay of the acellular pertussis vaccine components, pertussis toxoid, filamentous haemagglutinin and pertactin.

The development of acellular pertussis vaccines has raised a number of issues relevant to the control of these products. Of particular importance is the need for robust and accurate in vitro assays for the antigen content of the vaccines which might contain up to five different antigen components, each of which needs to be independently assayed. This paper describes a simple method for the quantification of three component antigens. Because relatively high doses of purified antigens are used in those preparations, the elimination of residual toxicity is a major concern. This is achieved by genetic modification of chemical treatment. The latter results in modification of the immunological reactivity of the antigens making direct assay by such methods as ELISA ineffective. A single radial diffusion technique using polyclonal antisera for the assay of pertussis toxoid (PTxd), chemically treated filamentous haemagglutinin (FHA) and pertactin (69 kDa) has been developed. The method uses low concentrations of antisera, allowing accurate and reproducible quantification of antigen content as low as 25 microg/ml of protein for pertussis toxoid and filamentous haemagglutinin and 5 microg/ml for pertactin. Since by the addition of detergent, diffusible subunits are produced irrespective of the original physical state of the antigens, the assay is suitable for assay of these antigens after detoxification/or stabilization by chemical treatment and is able to determine the differences between preparations which have the same protein concentration but different antigenic contents. This provides a means for assuring the consistency of the antigens after detoxification/or chemical stabilization which could be used as an in-process control method for acellular pertussis vaccines.

Assessing the significance of reverse transcriptase activity in chick cell-derived vaccines.

A recent publication reported the detection of low levels of the enzyme reverse transcriptase (RTase) in live viral vaccines prepared in chick embryo cells. The enzyme was detected using an assay with greatly increased sensitivity compared to more conventional methods. The authors have confirmed the observation of RTase activity and demonstrate that the activity is not dependent on the production of viral vaccines in chick cells but is present ubiquitously in chick embryonic fluids. The authors have also been unable to transmit the RTase activity from chick cells to a wide variety of cells of human, monkey, rabbit and turkey origin, suggesting that the activity is not associated with an avian agent capable of infecting these cells. It is concluded that the data available present no cause for concern over the safety of vaccines derived in chick cells and current WHO requirements for such vaccines remain appropriate.

Strategies for AIDS vaccines.

In the global AIDS epidemic, over half of all infections have occurred in people less than 25 years old resulting in profound social, economic and demographic consequences. Current estimates indicate that the present 15 million HIV infections will increase to over 30 million by the end of the millennium. For most countries a safe and effective vaccine offers the only hope of controlling the spread of this disease. The development of an effective vaccine against HIV is beset with formidable obstacles. Despite these difficulties, substantial progress has been made towards developing effective strategies for vaccination. Human clinical trials and animal models for AIDS, particularly simian immunodeficiency virus (SIV) infection of macaques, have proved invaluable in this quest. Inactivated virus vaccines induced potent protection in this model, but subsequent studies revealed that protection was mediated by antibody to cellular proteins present in the vaccine preparations and on the surface of infecting virions. This surprising observation has provided an alternative and complementary approach to the development of vaccines against HIV in man which is still being pursued. Live attenuated vaccines were initially dismissed as far too hazardous. However, the concept has recently been reexamined in the light of powerful evidence that attenuated SIV induces potent protection against a wide variety of viruses administered by intravenous or mucosal routes and even against challenge with viable virus-infected spleen cells. Efforts are now underway to understand the mechanism of this protection and to attempt to reproduce it by less hazardous means. Considerable effort has been devoted to the development of subunit HIV vaccines, predominantly based on the envelope glycoproteins of the virus. Extensive clinical trials in human volunteers have established that these vaccines are safe and antigenic. However, the immune responses appear to be transient and the antibodies induced do not neutralize the primary isolates of HIV which are circulating in the population. There are now three possible approaches to an AIDS vaccine which are being actively pursued.

Where are we now with vaccines against AIDS?

PIP: Extensive studies in experimental animal models and phase I and II clinical trials in humans provide some grounds for optimism about developing a vaccine for AIDS. Over 200 monkeys have been protected by simple inactivated vaccines against infection with a lethal challenge dose of simian immunodeficiency virus (SIV). Passive transfer of the antibody to SIV has been shown to prevent infection. Recent vaccination with attenuated, live SIV protected against a challenge with 1000 monkey infectious doses of virus. Studies in chimpanzees have been limited to the unrepresentative IIIB strain of HIV-1. Purified recombinant envelope protein either as the gp 120 surface unit or as the entire gp 160 protein has protected a proportion of chimpanzees against infection. Several experimental immunogens have been tested in phase I and II clinical trials in human volunteers. 5 different recombinant HIV envelope subunits, live recombinant vaccinia virus expressing HIV envelope, and synthetic peptides or virus-like particles derived from yeast, but expressing HIV core proteins, have been used for vaccines that have proved safe. However, large quantities of the subunit HIV antigens are required, and the humoral immune responses are short-lived. Trials with the live recombinant vaccinia virus expressing HIV envelope represent the first use of live recombinant virus in humans. Further studies are under way in France with an avian poxvirus vector, which is host restricted and therefore potentially safer. STudies with combinations of live recombinant virus vaccines followed by immunization with purified subunits indicate that this approach may stimulate both cellular immunity and neutralizing antibodies at higher concentrations than previously observed. The WHO has identified sites in Brazil, Rwanda, Thailand, and Uganda for large scale trials of the efficacy of AIDS vaccines that will probably begin within 5 years.^ieng

Direct isolation in eggs of influenza A (H1N1) and B viruses with haemagglutinins of different antigenic and amino acid composition.

Influenza A (H1N1) and influenza B viruses from clinical samples were isolated in the amniotic cavity of embryonated hens' eggs by classical techniques and propagated in the allantoic cavity. Virus progeny from different eggs which had been inoculated with virus material from the same clinical sample possessed antigenically distinguishable haemagglutinins (HAs). Virus progeny of some eggs possessed HAs which were serologically identical to those of virus isolated in parallel in mammalian (MDCK) cells. These egg-grown viruses possessing HAs with the antigenic phenotype of mammalian cell-grown viruses appeared to be antigenically related to epidemic influenza virus because post-infection human sera reacted to high titre with the virus HA. Specific nucleotide changes were detected in the HAs of the viruses isolated directly in eggs at positions 163 and 189 for influenza A (H1N1) viruses or positions 141 and 196 to 198 for influenza B viruses. Egg-isolated viruses which possessed the antigenic phenotype of mammalian cell-grown viruses retained glycosylation sites at positions 163 and 196. The viruses isolated directly in embryonated hens' eggs which possessed the HA antigenic phenotype and glycosylation sites of MDCK cell-grown virus can, unlike the latter viruses themselves, be used as candidate influenza vaccine viruses.

Studies on the immunogenicity of Chinese hamster ovary cell-derived recombinant gp120 (HIV-1IIIB).

Recombinant DNA-derived gp120 (HIV-1IIIB) expressed in chinese hamster ovary cells elicited specific humoral and cell-mediated immune responses in a variety of mammals. Antisera from immunized rabbits, sheep and goats recognized virus-derived gp120 and its precursor (gp160). Neutralizing antibodies were also elicited, but only in a few animals, and this may be related to the protein's susceptibility to cleavage through the neutralizing domain. However, in rabbits the degree of cleavage of gp120 had little or no effect on its antigenicity or immunogenicity. All antisera had limited cross-reactivity to envelope glycoproteins from a panel of HIV-1 isolates suggesting that immunodominant antibody epitopes are in variable regions of the recombinant gp120. Antigen-specific T-cell responses were detected in immunized macaques and were found to be stronger and more prolonged when gp120 was administered in Freund's adjuvant rather than alum.

A host-cell-selected variant of influenza B virus with a single nucleotide substitution in HA affecting a potential glycosylation site was attenuated in virulence for volunteers.

An influenza B virus was passaged in man (virus A) and then in human embryo trachea (C) and into embryonated eggs (D) or directly into eggs (B). Virus A, B, and C had the same (cell-like) haemagglutinin phenotype on reaction with selected monoclonal antibodies while D had an "egg-like" phenotype. The viruses were administered at a dose of 1,000 TCD50 (for MDCK cells) by intranasal inoculation to groups of 27 or 28 volunteers. Viruses A, B, and C all produced disease in six to eight volunteers, whereas D produced no illness and only four volunteers were infected. The viruses shed by the volunteers were indistinguishable from those with which they were inoculated. The haemagglutinin genes of the viruses were sequenced and changes were detected indicating amino acid substitutions at position 196-198 in the attenuated egg-grown virus D whereby a potential glycosylation site present in the other viruses was lost.

A collaborative study to establish the International Reference Reagent for hepatitis B vaccine containing plasma-derived hepatitis B surface antigen.

A collaborative study was conducted to establish a suitable international reference reagent for hepatitis B vaccine for use in immunogenicity assays. The limiting dilution required to induce antibodies in 50% of the test animals was determined for the proposed international reference reagent and three other plasma-derived hepatitis B vaccines. The minimum antigenic dose of these preparations varied widely (100-fold range) between laboratories. However, the expression of potencies of vaccines relative to the proposed International Reference Reagent reduced the variation between laboratories to within a 10-fold range. The reference reagent is intended for use in assays of hepatitis B vaccines in mouse (or guinea-pig) immunogenicity studies. For products made by different procedures, clinical trials in humans are necessary to establish a correlation between the immunogenic potency in animals and man.

Genetic basis of attenuation of the Sabin type 3 oral poliovirus vaccine.

The poliovirus type 3 Sabin oral poliovirus vaccine strain P3/Leon/12a1b differs in nucleotide sequence from its neurovirulent progenitor P3/Leon/37 by just 10 point mutations. The contribution of each mutation to the attenuation phenotype of the vaccine strain was determined by the construction of a series of recombinant viruses from infectious cDNA clones. The neurovirulence testing of recombinant viruses indicated that the attenuation phenotype is determined by just two point mutations: a C to U in the noncoding region at position 472 and a C to U at nucleotide 2034 which results in a serine-to-phenylalanine amino acid substitution in the structural protein VP3.

Medical Research Council AIDS Directed Program: program plan and research opportunities.

In 1987 the U.K. Medical Research Council established a Directed Programme of AIDS Research, the primary objective of which is the development of vaccines and therapeutic approaches against HIV infection and AIDS. The Programme's activities have been rapidly built up and by mid-1989 comprised some 130 individual research projects. The Programme provides the framework for a cohesive approach to AIDS research, comprising a clearly defined scientific development plan, the central provision of resources including laboratory reagents and specialized laboratory facilities and special arrangements for interaction with industry and for international collaboration and training. This article describes the organization, scientific strategies and progress of the Programme.