Antibody responses to antigenic determinants of influenza virus hemagglutinin. I. Thymus dependence of antibody formation and thymus independence of immunological memory.

Using immunodiffusion methods it has been shown that purified hemagglutinin (HA) extracted from two related strains of influenza A viruses (A/PR8/34 and A/FM1/47) have two distinct antigenic determinants, or groups of determinants. One determinant is cross-reactive while the other is strain-specific. Antisera raised in normal mice against HA were shown to contain two populations of antibody molecules, each directed against one of the determinants. Immunization of thymus-deprived (TXBM) mice showed a strong thymus dependence of antibody formation to HA. However, the thymus dependence of antibody formation against the cross-reactive determinant could be overcome by repeated inoculations of HA in TXBM mice, indicating a different handling of two portions of the same molecule by the immunological system. Strong, secondary-type responses to the strain-specific determinant were observed in primed thymus-deprived mice after reconstitution with virgin thymus cells, showing that specific immunological memory was elicited by this determinant despite the absence of detectable antibody secretion. These findings are interpreted as examples of immunological recognition and memory mediated by B lymphocytes and discussed in terms of mechanisms of T and B lymphocyte co-operation. It is suggested that the helper effect of T lymphocytes is exerted at a late stage in the differentiation of specific populations of B cells into antibody-secreting cells.

Antibody responses to antigenic determinants of influenza virus hemagglutinin. II. Original antigenic sin: a bone marrow-derived lymphocyte memory phenomenon modulated by thymus-derived lymphocytes.

Mice immunized sequentially with two related influenza virus hemagglutinins (HA) produced a secondary antibody response with two different specificities. Some antibodies were specific for determinants common to both HA's. Paradoxically, some antibodies were directed to determinants existing only in the HA first encountered. Primed spleen cells treated with anti-theta serum and complement were transferred from animals immunized with the first HA to either normal, irradiated, or thymus-deprived recipients. These memory cells were boosted in the recipients with either the homologous or the heterologous cross-reacting HA. B-memory lymphocytes were shown to be directly triggered by both HA's and to be able to secrete, independently of T lymphocytes, antibodies to both kinds of determinants. However, T cells were shown to modulate this secondary response by either enhancing or suppressing antibody secretion by B-memory cells, depending on experimental conditions. These results are discussed in terms of antigen recognition by B cells and of kinetics of development of immunological memory.

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.

Biochemical and serological studies of influenza B viruses: comparisons of historical and recent isolates.

The genetic characteristics of 24 representative influenza B viruses isolated in widely different geographical areas of the world between 1940 and 1980 were analysed using either RNA:RNA hybridisation or oligonucleotide mapping. Additional biochemical characterisation included electrophoretic analysis of virus-induced polypeptides and virion RNAs. A panel of monoclonal antibodies to virus HA was used to investigate serological relationships between the viruses. The influenza B viruses examined constituted a genetically and serologically related group but mutational changes were detected in all eight genes of the viruses isolated in different eras and also in genes of viruses isolated in the same epidemic year. Regardless of the overall and dominating similarities, at a higher level of discrimination it was clear that certain genetic and serological relationships were more complex than expected and, for example, some recently circulating field viruses were apparently more closely related antigenically and genetically to viruses isolated five to twelve years previously than to other viruses isolated concurrently. No evidence of recombination with hitherto undescribed influenza B viruses and with genes coding for internal proteins was detected.

Monoclonal antibodies which block cellular receptors of poliovirus.

The isolation and properties of monoclonal antibodies which specifically inhibit the binding of poliovirus types 1, 2 and 3 to cells are described. The antibodies were of the IgG class and blocked infection of cells by all strains of the three poliovirus serotypes, but by none of a wide range of other viruses examined, including nine human enteroviruses. The antibodies prevented poliovirus growth in all susceptible human and primate cells tested. We conclude that the antibodies are directed against the receptor site for poliovirus which is distinct from those required by other picornaviruses, and which seems to be antigenically well conserved between cells of human and primate origin.

A single-radial haemolysis technique for measurement of antibody to influenza virus neuraminidase in equine sera.

A modified single-radial-haemolysis (SRH) test for measurement of antibody to influenza virus neuraminidase (NA) is described. The test requires treatment of sheep erythrocytes with butanol to increase sensitivity. In comparative assays, SRH was found to be more sensitive than the conventional neuraminidase-inhibition test. The SRH test was reproducible, specifically measured antibody to influenza NA and was easy to use. SRH antibody responses in ponies vaccinated with bivalent equine influenza vaccine were shown to be vaccine dose-related but were lower in magnitude and shorter in duration than comparable anti-haemagglutinin responses.

Antibodies to poliovirus detected by immunoradiometric assay with a monoclonal antibody.

An immunoradiometric assay (IRMA) for the assay of antibodies to poliovirus antigens is described. Dilutions of the test sera or whole (finger prick) blood samples were incubated with the Poliovirus antigen bound to a solid phase and the specific antibody was detected by the addition of a mouse anti-human IgG monoclonal antibody (McAb), which was itself revealed by iodinated sheep IgG anti-mouse F(ab). We have shown that this technique is suitable for the estimation of IgG anti-poliovirus antibodies induced in children following polio vaccine. The present study shows that SPRIA provides a simple and inexpensive method for serological studies with poliovirus particularly for use in large-scale surveys.

Studies on the attenuation of the Sabin type 3 oral polio vaccine.

The genetic basis of attenuation of the poliovirus type 3 vaccine strain P3/Leon 12a1b has been investigated by comparing the nucleotide sequence of this strain with that of its neurovirulent progenitor P3/Leon/37 and by constructing recombinants between these two viruses using infectious cDNAs. Preliminary results suggest that attenuation is caused by just two point mutations, one occurring in the 5' non-coding region and the other causing an amino acid change in coat protein VP3.

An improved immunoblotting procedure for the detection of antibodies against HIV.

Immunoblotting ('Western blotting') is routinely used for detection of antibodies against HIV in the diagnosis of HIV infection. We describe an improved procedure, which does not require virus purification and is easy to control for 'false-positive' results. The technique also does not produce erroneous results due to reactivity of the developing system with residual cellular proteins or viral antigens and does not give high nonspecific background staining. The technique can be applied to the detection of antibodies to HIV in serum, plasma, and blood products.

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

The influenza virus: Antigenic composition and immune response.

The architecture and chemical composition of the influenza virus particle is described with particular reference to the protein constituents and their genetic control. The dominant role in infection of the surface proteins - haemagglutinins and neuraminidases - acting as antigens and undergoing variation in time known as antigenic drift and shift is explained. The immuno-diffusion technique has illuminated the interrelationships of the haemagglutinins of influenza A viruses recovered over long periods of time. The H0 and H1 haemagglutinins are now regarded as a single sub-type with H2 and H3 representing the haemagglutinins of the 1957 and 1968 sub-types. Animal influenza viruses of pigs, horses and birds are described. A relation to human influenza strains has been shown to exist in certain instances as is the capacity of some human strains to pass to the animal kingdom.

Antibodies to the strain-specific and cross-reactive determinants of the haemagglutinin of influenza H3N2 viruses. 1. Preparation of antibodies and in vitro studies.

The serological analysis of antibodies to the haemagglutinin (HA) of influenza A viruses of the Hong Kong (H3N2) subtype is described, using haemagglutination-inhibition, immuno-double-diffusion and single-radial-diffusion techniques. By cross-absorption of antisera to purified HA antigens, different populations of antibody molecules were obtained, which are designated strain-specific and cross-reactive and characterized in terms of their antigenic specificities for HA antigens of the homologous and antigenically variant H3N2 viruses. A narrowly strain-specific population of antibodies (SS"HK) was obtained as the residual antibody in antiserum to A/Hong Kong/1/68 HA after absorption with the closely related A/England/42/72 virus, whilst a contrasting broadly cross-reactive population (CR'HK) was obtained by absorption of the anti-A/Hong Kong/1/68 HA serum with the more distantly related strain A/Victoria/3/75 and eluting the cross-reactive antibodies from the absorbing virus. Similarly, specific and cross-reactive antibodies were derived from antiserum to A/Victoria/3/75 HA antigen by absorption with A/Hong Kong/1/68 virus. Single-radial-diffusion tests were performed, involving sequential application of different antibody preparations in the same wells in immunoplates containing intact virus particles. The cross-reactive and strain-specific antibodies differed in their property of mutual interference of attachment ot antigen. The results suggested that the cross-reactive antigenic determinants on the HA subunit may be located closer to the distal end of the molecule than the strain-specific determinants. Further tests employing single-radial-diffusion showed that there are more cross-reactive than strain-specific sites available for antibody in the intact virus particle. The strain-specific antibodies also gave higher haemagglutination-inhibition titres per microgram IgG than the cross-reactive antibodies.

Quantification of influenza virus structural proteins using rocket immunoelectrophoresis.

The major influenza A virus structural antigens, matrix protein, nucleoprotein, haemagglutinin and neuraminidase were measured rapidly and accurately using a rocket immunoelectrophoresis technique. Virus was disrupted with 1% (w/v) sodium sarcosyl and electrophoresed into agarose containing specific antiserum to the individual virus structural proteins in 0.05 M-barbitone buffer, pH 8.6, for 1 to 4 h. For haemagglutinin antigen assays statistical analysis indicated that the coefficient of variation within an immunoelectrophoresis plate was 8.0% for antigen concentrations in the range 15 to 40 microgram/ml protein. For haemagglutinin and matrix protein the method was sufficiently sensitive to measure concentrations of antigens as low as 1.5 and 2.0 microgram/ml respectively. By incorporation in the agarose of mixtures of antisera against specific antigens of the virus, haemagglutinin, matrix or nucleoprotein could be assayed simultaneously.