G. L. Ada

Expression of Cytokines by Recombinant Vaccinia Viruses: A Model for Studying Cytokines in Virus Infections in vivo

Host defence against virus infection occurs in three distinct but sometimes interacting phases: innate resistance that is non-inducible and immediate; an early, inducible phase that is largely antigen non-specific; and a relatively late T celldependent phase that is inducible, highly antigen-specific and generates immunological memory (Janeway 1988, 1989). Natural killer (NK) cells and macrophages are active participants in the innate and early, interferon-inducible phases of the response (reviewed in Welsh 1986) and begin to exert their antiviral function before the generation of antigen-specific immune responses, namely antibody and cytotoxic T cells (CTL). The latter are generally believed to be crucial in the control of and recovery from most primary viral infections (Blanden 1971a, b, 1974).

Purification and properties of neuraminidase from Vibrio cholerae.

SUMMARY: A method is described for the purification of neuraminidase from culture fluids of Vibrio cholerae. Five steps are involved: fractionation with methanol, adsorption to and elution from human red cells, fractionation with ammonium sulphate, chromatography on columns of hydroxyl apatite, crystallization. From 35 l. of culture filtrate, an average yield of 21% of the original enzyme activity was obtained as crystals. The degree of purification was about 5000 fold. Purified neuraminidase possessed 12·6 × 106 units of biological activity/mg. dry weight, and gave a value for E 1% 280 mμ of 8·96, measured at 0·025% (w/v). Enzyme activity was stimulated by calcium ions and inhibited by ethylenediaminetetra-acetate. In the presence of 0·001 M-CaCl2, neuraminidase showed maximum activity at pH5·6. With sialyl lactose as substrate, a value of 1·2 x 10-3M was found for the Michaelis constant. At an enzyme concentration of 0·16 μg./ml., V max. was 0·021 μM N-acetylneuraminic acid/min./ml. The enzyme was stable when dried from the frozen state and stored under vacuum at 0°. A suspension of crystals in water also retained activity when stored at 0°. Solutions of crystalline neuraminidase showed a small increase in activity when stored at 0° for several weeks. This effect was greatest at pH 6·7 and 8·5 but was barely detectable at pH 4·6. At pH 5·6 or 6·7, the enzyme lost about 20% of its activity over a period of 2 hr. at 37° (concentration = 15 μg./ml.) No proteolytic activity nor N-acetylneuraminic acid aldolase activity was detected in the crystalline preparation.

An Analysis of Effector T Cell Generation and Function in Mice Exposed to Influenza A or Sendai Viruses

The activation and effector function of T cells are governed by a dual specificity of recognition system involving, on the one hand, a foreign antigen such as a hapten, a soluble protein molecule or a viral antigen; and, on the other hand, a self antigen coded for by the major histocompatibility complex (MHC). According to the H-2 region involved, mouse T lymphocytes can be subdivided into two subsets T cells which recognize antigen in association with I region MHC products and T cells which recognize antigen associated with K,D region MHC products. The H-2 restriction pattern and the Lyt phenotype of T lymphocytes are closely linked: I region restricted T cells are Lyt 1*, 2 ,3 ; K,D region restricted T cells have the Lyt T, 2*, 3* phenotype. In the latter case, it may be that the observed correlation reflects participation of Lyt 2, 3 antigens in the structure of the T cell receptor (Hollander et al. 1980, Sarmiento et al. 1980). T cells of both these subsets take part in a variety of functions. It has been proposed, and we would support the concept, that T cells evolved as part ofthe defence system to infectious diseases, and viral infections may have played a particularly important role. Thus, infection of cells by many different viruses results in the early expression of viral antigens at the surface of infected cells.

Cytotoxic T Cells in the Lungs of Mice Infected with an Influenza A Virus

Cytotoxic T cells are present in the lungs and the bronchoalveolar washings of mice infected intravenously (i.v.) or intranasally (i.n.) with live influenza A/WSN virus. After i.v. injection, cytotoxic T cell activity in both spleens and lungs reaches a peak at 6 days when the level of infectious virus recovered from the lungs falls sharply and the mice do not die. If a lethal dose of virus is given intranasally, very high levels of virus appear rapidly in the lungs, and the development of lung consolidation follows slightly behind the appearance of cytotoxic T cells there When a non‐lethal dose of virus is given intranasally, lower levels of virus are found in the lung and the appearance of cytotoxic T cells is delayed. These results suggest that the cytotoxic T cells play a protective role if the level of virus in the lungs does not reach very high levels. After injection of antithymocyte serum, the subsequent level of cytotoxic T cell activity in the lungs was greatly reduced, suggesting that the T cells recovered in lungs had at an earlier stage been circulating cells. However, splenectomized mice develop high levels of cytotoxic T cell activity, after intranasal infection of mice, indicating that the spleen did not contribute substantially to the T cells recovered in the lungs.

Cellular Immune Responses in the Murine Lung to Local Immunization with Influenza A Virus Glycoproteins in Micelles and Immunostimulatory Complexes (Iscoms)

Primary immunization with a single inoculum of either micelles or iscoms containing influenza A virus glycoproteins failed to induce either B or cytotoxic T (Tc) cell responses. In contrast, immunization with two inocula of iscoms, but not micelles, resulted in the appearance of influenza virus‐specific antibody‐secreting cells (ASC) but not Tc cells in the lung. There was a 10‐fold increase in Tc cell precursor frequency and an increase in ASC generated by secondary in vitro stimulation of lung cell cultures obtained from mice primed with iscoms but not micelles. In mice primed with infectious virus, secondary immunization with either micelles or iscoms increased the number of ASC in the lung and elicited virus‐specific Tc cell responses. In contrast homologous virus challenge failed to induce detectable secondary B or Tc cell responses.

The Roles of Influenza Virus Haemagglutinin and Nueleoprotein in Protection: Analysis Using Vaccinia Virus Recombinants

Vaccinia virus recombinants expressing haemagglutinin (HA) or nucleoprotein (NP) from influenza virus A/PR/8/4 were used to investigate protective immunity in mice, with two protocols. Protection was assessed by mortality and morbidity rates and by lung virus titres after infection intranasally with A/PR/8/34. In the first protocol, mice immunized with vaccinia‐HA recombinaant virus and infected intranasally with A/PR/8/34 were almost totally protected, but mice immunized with vaccinia‐NP virus were very poorly protected. In the second protocol, the recombinant viruses were used to stimulate in vitro T cells that are specific for HA and NP; both populations of T cells, when transferred to A/PR/8/34‐infected mice, afforded good protection. The results indicate that an immune response specific for just HA provided protection that was almsot indistinguishable from that provided by whole A/PR/8/34. On the other hand, immunization with vaccinia‐NP provided poor protective immunity, despite the fact that transferred NP‐specific T cells were very effective and vaccinia‐NP immunization has previously been shown to stimulate cytotoxic T cells. These results demonstrate that a single viral antigen, delivered by live vaccinia virus, can provide effective protection, but that immunization for cross‐protection against heterologous influenza virus remains elusive.

Cell-mediated immune responses to influenza virus antigens expressed by vaccinia virus recombinants

Recombinant vaccinia viruses enable studies of immune recognition of antigens expressed from single viral genes. We have constructed recombinants expressing the haemagglutinin (HA) and nucleoprotein (NP) genes of the influenza virus A/PR/8/34 (H1N1). These recombinant viruses together with a recombinant expressing the HA from influenza virus A/JAP/305/57 (H2N2) have been used to examine the cytotoxic T lymphocyte (CTL) response to these influenza virus antigens. Both antigens are recognised by murine CTL and recognition of HA is influenza virus subtype-specific, whereas recognition of NP is crossreactive. In limiting dilution studies approximately 10% of the influenza CTL response is HA-specific, while approximately 30% of the response is NP-specific. Despite the ability of NP to stimulate a significant CTL response, mice immunised with the NP-vaccinia recombinant are not as well protected from subsequent lethal challenge with influenza virus, as mice immunised with the HA vaccinia recombinant. These studies demonstrate that viral antigens expressed from vaccine recombinants can provide protective immunity and that the influenza-poxvirus recombinants can provide data on protective immunity generated by individual viral proteins.

The Recovery of Mice from Influenza Virus Infection: Adoptive Transfer of Immunity with Immune T Lymphocytes

Transfer of primary or secondary influenza‐immune spleen cells to mice infected intranasally with influenza virus resulted in a significant clearance of virus from the lungs and the protection of the recipients from death. The antiviral activity was associated only with intact, viable cells and was not due to carryover of virus. The effector cell population responsible for the antiviral effect was shown to be T cells. Thus, the removal of adherent, phagocytic and Ig+ cells did not affect the antiviral activity, whereas it was destroyed with antitheta serum and complement. Antiviral activity was specific and was best expressed if the virus used to infect the recipients and to generate immune cells was the same strain. Further work will be necessary to define rigorously the role of different viral antigens in cell‐mediated immune response to influenza virus infection.

Influenza Virus Nucleic Acid: Relationship between Biological Characteristics of the Virus Particle and Properties of the Nucleic Acid.

SUMMARY: Five A strains and three B strains of influenza virus were purified and found to contain ribonucleic acid in amounts varying from 0·75 to 1·1 %. The proportion of the purine and pyrimidine derivatives in the nucleic acid of each strain was determined. When the ratio adenine + uracil: guanine + cytosine was evaluated, the following values were obtained: A strains —PR8, 1·27± 0·02; MEL, 1·22 ± 0·01; WSE, 1·26 ± 0·01; SWINE, 1·24 ± 0·04; CAM, 1·28 ± 0·01. B strains—LEE, 1·42 ± 0·04; MIL, 17·43 ± 0·05; ROB, 1·38 ± 0·01. The nucleic acid content of PR8 virus preparations of varying degrees of incompleteness was determined both by specific absorption at 260 mμ. and by estimation of phosphorus present in the nucleic acid extract. As measured by both methods, virus preparations of low infectivity were found to have a decreased nucleic acid content. The demonstration of specific differences in the nucleic acid of A and B strains and of the relationship between the infectivity and nucleic acid content of the virus affords strong evidence that the nucleic acid is an intrinsic part of the influenza virus particle.

The Recovery of Mice from Influenza A Virus Infection: Adoptive Transfer of Immunity with Influenza Virus‐specific Cytotoxic T Lymphocytes Recognizing a Common Virion Antigen

Mice inoculated intranasally with infectious influenza virus of a given A strain were adoptively transferred 24 h later with preparations of secondary influenza virus‐immune T cells generated either in vitro or entirely in vivo. The immune cells were raised during infection with homologous or heterologous A strain influenza viruses or with a type B virus. The greatest antiviral effect, measured by reduction in lung virus level of recipient mice, occurred if homologous viruses were used. Sharing of haemagglutinin specificity was shown to be important, but significant antiviral activity was still expressed if neither haemagglutinin nor neuraminidase antigenic specificities were shared. The antiviral effect was type‐specific. Adoptive transfer of type A influenza immune T cells did not express antiviral activity against type B virus, and vice versa. On the basis of earlier work, the effector population in the transferred cells was cytotoxic T cells (Tc). Intranasal reinfection of mice with a heterologous type A virus sharing neither haemagglutinin nor neuraminidase antigenic specificity with the first infecting virus induced enhanced and earlier production of cross‐reactive Tc against type A influenza viruses. This was paralleled by significantly lower virus levels in the lungs. The results of this work demonstrate heterotypic cell‐mediated immunity in influenza virus infection in mice.