Live Virus Challenge Research Articles

Long-Term Maintenance of B Cell Immunity to Influenza Virus Hemagglutinin in Mice Following DNA-Based Immunization

This study demonstrates that gene-gun inoculation of mice with DNA encoding the influenza virus hemagglutinin (HA) results in the life-long maintenance of protective B cell responses. Using a sensitive single-cell enzyme-linked immunospot assay, we show that all of the HA-specific plasma cells are localized in the bone marrow and spleen 1 year postimmunization. As a consequence of prior virus challenge, only a small population of antibody-forming cells was found in the lymphoid tissues associated with the respiratory tract. The tissue distribution of HA-specific plasma cells in these mice was identical to the profile in infected controls. Complete protection against live virus challenge in the aged vaccinated mice did not require prior exposure to virus. Thus, immunization with the DNA vaccine provides long-term protective immunity against otherwise lethal infection.

Protection of macaques against simian immunodeficiency virus infection with inactivated vaccines: comparison of adjuvants, doses and challenge viruses

Nine European laboratories contributed a total of 98 macaques towards a collaborative trial to study the ability of formaldehyde-inactivated or subunit SIV vaccines to protect immunized animals against live virus challenges. Four adjuvants, three dose levels and two immunization schedules were compared. Fifty-two of 61 (85%) immunized animals were protected against infection after challenge with either homologous or heterologous virus strains grown in human cells. Optimum protection required a high dose of antigen and a prolonged immunization schedule. On the day of challenge the titres of antibodies to SIV and to host cell components, as well as the titres of neutralizing antibodies, were significantly higher in the protected animals than in the non-protected. Forty-four vaccinated macaques (of which 36 were protected against previous challenges grown in human cells) and 28 naive animals were then challenged with extracellular or cell-associated SIV grown in simian cells. All naive animals and all vaccinees challenged with extracellular SIV became infected. Four of the eight animals challenged with cell-associated viruses were protected. These results clearly indicate that vaccines which potently protect against SIV grown in human cells, do not protect against SIV grown in simian cells. The cell substrate on which challenge viruses are grown is clearly significant in interpreting the results of vaccine trials. This trial has demonstrated that SIV vaccines using different adjuvants can protect macaques against SIV grown in human cells but not against extracellular SIV grown in simian cells. These results have important relevance to the development of HIV vaccines for humans. The results also demonstrate the feasibility and value of large-scale European collaborative experiments in which scarce and ethically sensitive primate resources can be used to evaluate a wide range of vaccine variables.

Defective interfering type A equine influenza virus (H3N8) protects mice from morbidity and mortality caused by homologous and heterologous subtypes of influenza A virus.

Intranasal administration of defective interfering A/equine/Newmarket/7339/79 (H3N8) influenza virus (DI EQV) protected mice from otherwise lethal intranasal infection with homologous virus (EQV) or with the heterologous subtypes A/WSN (H1N1) or A/PR/8/34 (H1N1). Such protected mice showed little or no sign of clinical disease. Disease with only low mortality resulting from a 'low' dose of WSN was completely prevented with a 100-fold lower dose of DI EQV (4 haemagglutinating units/ml or 12 ng virus/mouse), indicating that there was a roughly proportional relationship between the protective dose of DI virus and the infecting inoculum. DI EQV-protected mice continued to gain weight at the normal rate, whereas those treated with inactivated DI EQV ceased putting on weight for about 7 days and were still underweight nearly 3 weeks later. Unlike DI WSN, DI EQV inhibited multiplication of infectious WSN in the lungs by 20 to 60-fold. Intranasal DI EQV on its own gave little protection to mice challenged 24 days later with EQV suggesting that it was only weakly immunogenic. DI EQV afforded significant protection when given up to 5 days before live virus challenge indicating that the DI genome remained active in the respiratory tract for this period of time.

Anti-IL-4 treatment at immunization modulates cytokine expression, reduces illness, and increases cytotoxic T lymphocyte activity in mice challenged with respiratory syncytial virus.

Upon respiratory syncytial virus (RSV) challenge, mice previously immunized intramuscularly with inactivated whole virus express a Th2-like pattern of cytokine mRNA, while mice immunized with live virus intranasally express a Th1-like pattern. In this study, we evaluated the effects of anti-IL-4 treatment on the induction of immune responses after immunization. Mice treated with anti-IL-4 at the time of immunization with inactivated RSV had reduced clinical illness after live virus challenge, as measured by weight loss, illness score, and virus replication. This was associated with an augmented CD8+ cytotoxic T lymphocyte (CTL) activity, increased expression of IFN-gamma mRNA relative to IL-4 mRNA, and a higher titer of RSV-specific IgG2a in the anti-IL-4 treated mice before challenge. Anti-IL-4 administration at the time of challenge had no effects on illness, immunoglobulin isotype, or cytokine patterns. These results suggest that inhibition of IL-4 action at immunization can shift the selective activation of lymphocytes to a more Th1-like response. This cytokine milieu is associated with augmented CTL activity, which may be the factor responsible for rapid viral clearance and reduced illness at the time of remote RSV challenge.

Application of the immune complex for immune protection against viral disease

An immune complex (IC), composed of antigenic subunits of the Newcastle disease virus (NDV) and specific polyclonal allogeneic antibodies, was used to protect chickens against NDV. Antibodies in the IC were chicken immunoglobulin G. The antibody; antigen ratio in IC was 2.03. The IC was prepared at equivalence by direct mixing of NDV-infected allantoic fluid, treated with Triton X-100, and chicken anti-NDV serum. In order to bind NDV antigenic subunits to specific antibodies, previous isolation and purification of antigen is not required. Chickens were immunized with 1 mg IC, containing 0.3788 mg of viral antigens. The IC, prepared in the form of an oil-emulsion, was administered intramuscularly. The IC generated high levels of anti-NDV antibodies and successfully protected chickens against live virus challenge. Therefore, the IC could be recommended as a safe and environmentally convenient vaccine.

Protein of macaques against infection with simian type D retrovirus (SRV-1) by immunization with recombinant vaccinia virus expressing the envelope glycoproteins of either SRV-1 or Mason-Pfizer monkey virus (SRV-3)

Rhesus macaques were immunized with live vaccinia virus recombinants expressing the envelope glycoproteins (gp70 and gp22) of simian type D retrovirus (SRV), serotype 1 or 3. All of the animals immunized with either the SRV-1 env or the SRV-3 env vaccinia virus recombinant developed neutralizing antibodies against the homologous SRV. In addition, both groups developed cross-reactive antibodies and were protected against an intravenous live-virus challenge with SRV-1. The four control animals immunized with a vaccinia virus recombinant expressing the G protein of respiratory syncytial virus were not protected against the same SRV-1 challenge. Although SRV-1 and SRV-3 immune sera showed cross-neutralization, they failed to neutralize a separate, more distantly related serotype, SRV-2, in an in vitro assay. These findings are consistent with the known degree of serologic and genetic relatedness of these three SRV strains.

Responses of mice to murine coronavirus immunization

SummaryOral and/or intranasal inoculation of susceptible mouse genotypes with the JHM strain of mouse hepatitis virus (MHV-JHM) consistently results in T cell dysfunction as reflected by in vitro proliferative responses to mitogens or allogeneic cells. One approach to examining the mechanism responsible for the observed functional T cell suppression is to determine whether virus replication is required for its induction. To this end, mice were inoculated oronasally with MHV-JHM that was inactivated with short-wave ultraviolet light, betapropiolactone or psoralen. Mice were also inoculated with live MHV-JHM after recovery from homotypic or heterotypic MHV infection. Spleen cells from BALB mice inoculated oronasally with inactivated MHV-JHM yielded extremely variable in vitro proliferative responses after concanavalin A stimulation. MHV-susceptible mice exposed oronasally or intraperitoneally to virus inactivated by any of the minimum effective treatments failed to seroconvert. Immunization with psoralen-treated virus intraperitoneally in Freund's complete adjuvant or oronasally failed to protect from live virus challenge, but survivors had elevated virus-specific serum IgG antibody titers compared to mock-immunized controls at two weeks post-challenge. Spleen cells from mice that were challenged after recovery from homotypic live virus infection did not exhibit the profound in vitro T cell suppression normally observed during the acute stage of primary infection. In contrast, MHV-JHM challenge of mice vaccinated with heterotypic live MHV-S resulted in significantly depressed in vitro T cell function. The combined data suggest that either virus replication or exposure to more concentrated antigen may be required for induction of the dramatic T cell dysfunction that occurs as a consequence of MHV-JHM infection as well as for a detectable MHV-specific humoral response.

Worldwide survey of AIDS vaccine challenge studies in nonhuman primates: Vaccines associated with active and passive immune protection from live virus challenge

An AIDS Vaccine Surveillance System (AVSS) was designed and implemented to track the rapidly growing international database supporting the development of promising AIDS vaccines. Both preclinical nonhuman primate (NHP) and clinical human trials are tracked by the AVSS. This report presents summary data generated from the AVSS on the NHP AIDS vaccine/live virus challenge studies only. Summary data on more than 100 preclinical HIV/SIV vaccines are presented within the framework of 1) 13 arbitrary Vaccine Types, 2) studies grouped by animal model (i.e., chimpanzee/HIV-1, and macaque/SIV, HIV-2), and 3) immunization approach (i.e., active and passive). Systematic and timely presentations of these summary data, both here and in future reports, aim to promote a clearer understanding of both earlier and more recent preclinical AIDS vaccine developments.

Comparative analysis of the immunostimulatory properties of different adjuvants on the immunogenicity of a prototype parainfluenza virus type 3 subunit vaccine

The immunogenicity of a parainfluenza virus type 3 (PIV-3) subunit vaccine consisting of affinity-purified haemagglutinin—neuraminidase (HN) and fusion (F) surface glycoproteins was tested in guinea-pigs and hamsters. The ability of several different immunopotentiating agents to enhance the antibody response of animals to the PIV-3 surface glycoproteins was evaluated. The immunity induced by HN and F alone was compared with the response elicited by purified proteins combined with Freund's complete adjuvant, aluminium phosphate, Syntex's threonyl—muramyl dipeptide (MDP) SAF-MF formulation, or Ribi's adjuvant formulation containing BCG cell wall skeleton (CWS), trehalose dimycolate (TDM) and monophosphoryl lipid A (MPL) in a 2% squalene-in-water emulsion. Purified proteins were also incorporated into three different liposome formulations prepared by the detergent dialysis procedure. Immunization of guinea-pigs and hamsters with two 15 μg doses of the PIV-3 surface glycoproteins administered in the absence of adjuvant elicited high haemagglutination inhibition, neutralization and anti-fusion titres. The liposome preparations failed to enhance the antibody titres. Ribi's adjuvant formulation was effective at inducing a good secondary response to the purified proteins while the immunostimulatory effects of aluminium phosphate, Syntex and Freund's adjuvants were clearly demonstrated in both primary and secondary responses. When administered without adjuvant, a 15 μg dose of the HN and F mixture was capable of protecting hamsters against live virus challenge. The immunoprotective dose of the purified proteins could be reduced to at least 0.1 μg by the addition of aluminium phosphate, Syntex or Freund's adjuvants.

Initiation of cytotoxic T-cell response and protection of Balb/c mice by vaccination with an experimental ISCOMs respiratory syncytial virus subunit vaccine.

Respiratory syncytial virus is an important human pathogen causing serious lower respiratory tract infections of children and elderly people. Previous studies on the development of experimental subunit vaccines either expressed by recombinant DNA technology or prepared from purified viral proteins absorbed on adjuvant (ISCOMs) have shown promise. The present work reports on the effectiveness of an experimental ISCOMs vaccine in initiating humoral and cell-mediated immune responses and in providing overall protection upon live virus challenge in Balb/c mice; results indicate that vaccination by the intramuscular route is more effective, even if vaccination by the intranasal route also significantly reduced virus shedding.

Protection of BALE/c mice from respiratory syncytial virus infection by immunization with a synthetic peptide derived from the G glycoprotein

A synthetic peptide homologous to amino acids 174–187 of the G glycoprotein of the A2 strain of human respiratory syncytial (RS) virus (G/174-187) was shown to induce protection from live virus challenge of BALB/c mice after immunization with three doses of 50 μg of peptide coupled to keyhole limpet hemocyanin. Immunized mice showed high levels of circulating RS-specific antibodies as detected by ELISA assay; however, no neutralizing antibodies were found. Moreover, an important short-term cytotoxic T-cell response was observed with lymphocytes isolated from the lungs but not from the spleen of immunized mice. This response was lost 24 weeks after immunization; however, mice remained protected against challenge with live RS virus. In addition, a monoclonal antibody that specifically binds to peptide G/174-187 was found efficient in conferring passive protection from challenge: this data further supports our results on the importance of the 174–187 region in protection. Another peptide, spanning amino acids 144 to 159, was shown to induce neutralizing antibodies but did not confer protection.

Efficacy studies on a canarypox-rabies recombinant virus

Recombinant avipox viruses have been developed expressing the rabies glycoprotein gene. A fowlpox-rabies recombinant has previously been shown to be protective against live rabies virus challenge in a number of non-avian species 1. This report describes the development of a canarypox-rabies recombinant. A comparison is made of the protective efficacy of this recombinant with other pox-rabies recombinants.

Protective cellular retroviral immunity requires both CD4+ and CD8+ immune T cells

We have found previously that postexposure chemoprophylaxis with 3'-azido-3'-deoxythymidine (also known as zidovudine or AZT) in combination with recombinant human alpha A/D interferon fully protected mice exposed to a lethal dose of Rauscher murine leukemia virus (RLV) against viremia and disease. After cessation of therapy, over 90% of these mice were able to resist rechallenge with live RLV, thus demonstrating an acquired immunity. Adoptive cell transfer of 4 x 10(7) cells from immunized mice fully protected naive recipients from viremia and splenomegaly after RLV challenge. However, when these immune T cells were fractionated into CD4+ and CD8+ subpopulations, only partial protection was found when 4 x 10(7) T cells of either subset were given. Full protection against RLV challenge was seen again when the T-cell subsets from immunized mice were recombined and transferred at the same number into naive mice. We conclude that cellular immunity alone is protective and that both CD4+ and CD8+ cell types are required for conferring full protection against live virus challenge.

Preliminary report: protection of cynomolgus macaques against simian immunodeficiency virus by fixed infected-cell vaccine

Cynomolgus macaques were vaccinated with inactivated simian immunodeficiency virus-infected cells and 'Quil-A' as adjuvant at 0,4,8, and 36 weeks or at 0, 4, 8, and 16 weeks. 2 weeks later these animals, together with a similar unvaccinated group, were challenged with 10 MID 50 (50% monkey infectious doses) of a pool of SIVmac251 previously titrated in vivo. Virus was repeatedly isolated from unvaccinated animals on at least five separate occasions and proviral DNA was detected in circulating lymphocytes by polymerase chain reaction amplification. By contrast, virus and proviral DNA were not found in any of the vaccinated animals. However, the same vaccination regimen used after live virus challenge did not eliminate virus from previously infected macaques.

Virus envelope-based peptide vaccines against virus-induced mammary tumors

Previous studies by us and others established that mammary tumors induced by murine mammary tumor virus (MuMTV) could be prevented to various extents by prior vaccination with MuMTV-containing or subviral component immunogens. In this report, four predicted surface-accessible peptide regions (EP-1 to EP-4) of the major viral envelope glycoprotein (gp52) of C3H-MuMTV were tested as carrier-conjugated vaccines for the protection of Balb/c mice against a live virus challenge. With tumor incidence as an endpoint, vaccination with one of these synthetic peptides (EP-3) resulted in a significant reduction in the frequency of early onset tumors and 67% of the test animals remained tumor-free for the entire observation period (16 months). In contrast, only marginal protection was obtained by immunization with the intact glycoprotein (gp52). Immunologic interference may explain the lower protective efficacy of gp52, as compared to EP-3.

Complement-mediated, infection-enhancing antibodies in plasma from vaccinated macaques before and after inoculation with live simian immunodeficiency virus

Rhesus monkeys vaccinated against infection with simian immunodeficiency virus (SIV) were examined, in retrospect, for the presence of infection-enhancing antibodies and possible consequences associated with the presence of these antibodies. At the time of experimental inoculation with live virus, complement-mediated, infection-enhancing antibodies were detected in plasma specimens from six of six animals vaccinated with detergent-inactivated whole virus, from nine of nine animals vaccinated with Formalin-inactivated whole virus, and from seven of eight animals immunized with two SIV subunit preparations. The presence of infection-enhancing antibodies at the time of viral challenge gave no indication of predicting vaccine success or failure. After live-virus challenge, titers of infection-enhancing antibodies, like enzyme-linked immunosorbent assay titers, increased in unprotected animals and decreased or became undetectable in animals protected by vaccination. Thus, vaccine protection against SIV infection can still be achieved in the presence of detectable complement-mediated, infection-enhancing antibodies.

Vaccine protection against simian immunodeficiency virus infection.

Rhesus monkeys were immunized by multiple inoculations with purified, disrupted, noninfectious simian immunodeficiency virus (SIV) in adjuvant. Immunized monkeys developed anti-SIV antibodies detectable by whole-virus ELISA and by immunoblot reactivity; these antibodies had weak neutralizing activity. One week after the last immunization, monkeys were challenged with 200-1000 animal infectious doses of uncloned, live SIV. The same strain of SIV that was used for vaccination was also used for challenge. Anamnestic antibody responses and SIV recovery from peripheral blood were used to evaluate infection following the live virus challenge; two of six vaccinated monkeys showed no evidence of infection following the live virus challenge. Transfusion of 10 ml of whole blood from these two into uninfected, naive rhesus monkeys did not result infection of the recipients, providing further support for the lack of infection in the two previously vaccinated animals. Four of four unvaccinated control monkeys inoculated with these doses of live SIV became infected and three of these died with AIDS 118-258 days after infection. Only one of the six vaccinated monkeys has died to date. In situ hybridization with lymph node biopsy specimens suggested that the virus load was much higher in control macaques than in vaccinated macaques. These results indicate that vaccination with inactivated whole virus can protect macaques against challenge with live SIV. Furthermore, they provide hope that vaccine protection against human AIDS virus infection may be possible.

Recombinant fowlpox virus inducing protective immunity in non-avian species.

The natural host of fowlpox virus is limited to avian species. When inoculated into non-avian tissue culture cells, however, fowlpox virus can initiate an abortive infection. A fowlpox virus was engineered to express rabies virus glycoprotein. On inoculation of the recombinant virus into either avian (permissive) or non-avian (non-permissive) cells, the rabies glycoprotein was expressed as a membrane-associated antigen. Inoculation of the fowlpox virus recombinant into six different species of mammal resulted in specific immune responses to both fowlpox antigens and to rabies glycoprotein. In mice, cats and dogs the immune response was sufficient to protect against a live rabies virus challenge. The results demonstrate the utility of a fowlpox virus vector in immunizing non-avian species against rabies in the absence of productive viral replication of the fowlpox vector.

Sequential infection or immunization of ferrets with a series of influenza A (H3N2) strains (report to the Medical Research Council's Sub-Committee on influenza Vaccines (CDVIP/IV)).

Previous studies of boys at Christ's Hospital school have indicated that annual immunization with influenza virus vaccines did not significantly reduce the total incidence of influenza infection compared to unimmunized subjects. In view of the implications of this result, a similar study was conducted in ferrets to clarify these findings. Groups of ferrets were immunized or infected with a series of influenza A (H3N2) viruses over an 18-month period, and the immunity to subsequent live virus challenge was measured after each virus or vaccine exposure. The results indicated that live virus infection gave a more solid immunity than immunization with inactivated vaccine; and the serum haemagglutination-inhibiting antibody response was greater following immunization than following infection. In addition, differences in immunity could not be explained by measurements of cross-reacting and specific antibody, since the incidence of these antibodies was similar in both infected and immunized animals. The results do not suggest an explanation for the different levels of immunity induced following infection or immunization or the results obtained from the Christ's Hospital study. However, the relative contribution of various immune responses to virus or virus antigen is discussed, and it is suggested that the difference in immunity may lie in the ability of live virus infection to stimulate local antibody.

Human respiratory syncytial virus glycoprotein G expressed from a recombinant vaccinia virus vector protects mice against live-virus challenge.

Recombinant vaccinia virus vectors were constructed which expressed the major surface glycoprotein G of human respiratory syncytial (RS) virus. The biological activity of the G protein expressed from these vectors was assayed. Inoculation of rabbits with live recombinant virus induced high titers of antibody which specifically immunoprecipitated RS virus G protein and was capable of neutralizing RS virus infectivity. Immunization of mice by either the intranasal or the intraperitoneal route with recombinant virus that expressed only the G protein resulted in complete protection of the lower respiratory tract upon subsequent challenge with live RS virus.