Formalin-inactivated Vaccine Research Articles

Immunoprophylaxis and the control of respiratory syncytial virus disease.

Respiratory syncytial virus (RSV) remains one of the most important infectious causes of hospitalization in infants and children. This enveloped, RNA virus produces predictable yearly outbreaks of disease that typically peak between January and February in countries in the northern hemisphere.1 The outcome of RSV infection varies from mild upper respiratory tract infection in approximately 75% of infected infants and young children to severe life-threatening disease in a small percent of infected patients.2 In the United States, RSV lower respiratory tract infection accounts for nearly 50% of hospitalizations due to bronchiolitis and 25% of hospitalizations due to pneumonia.1 Serologic surveys suggest that by 2 years of age, more than 90% of all children have been infected by RSV.3 Whether RSV infection early in life predisposes to subsequent reactive airway disease remains an unanswered question. Reinfection is common, indicating that immunity to RSV following natural infection is less than complete.4-6 RSV lower respiratory tract disease occurs primarily in infants under 2 years of age; most infants who require hospitalization are previously healthy infants less than 6 months of age. Premature infants, infants born with congenital heart disease, and those with chronic lung disease (such as bronchopulmonary dysplasia [BPD]) constitute additional high-risk groups with high rates of hospitalization due to RSV infection.7-13 A recent report describes RSV mortality rates among such hospitalized infants of 4% to 5%.14Pre-engraftment bone marrow transplant recipients, solid organ transplant recipients, and lymphopenic children receiving chemotherapy appear to suffer even higher mortality rates, although prospective data are not available.15 Morbidity in these groups is also great; the average hospital stay and intensity of care for such children may be several times that of previously healthy infants. Despite the importance of RSV as a pathogen in the pediatric age group, …

RATIONALE FOR ANTIBODY PROPHYLAXIS OF RSV INFECTIONS 18

In the wake of failed clinical trials of a formalin-inactivated RSV vaccine in the 1960's, the trial's sponsors, noting that the vaccinees who had experienced enhanced RSV disease had moderate amounts of vaccine-induced antibody, concluded erroneously that antibody to RSV was harmful. This hypothesis was further justified by the observation that, in unvaccinated patients, the most serious cases of RSV disease were seen in infants under 6 months of age, a time when maternal antibody is still present. For nearly 15 years this hypothesis, which had not been subjected to experimental verification due to the lack of an animal model of RSV infection, remained conventional wisdom. The development of the cotton rat model of RSV infection provided the opportunity to examine the nature of the immune response to RSV. In a crucial experiment involving the parabiotic linking of an immune to a non-immune cotton rat, we found that complete resistance to pulmonary RSV infection was conferred to the non-immune partner via whole blood. Subsequent blood fractionation experiments showed that the protective factor resided in the immune globulins, and that the cellular component of the blood neither conferred protection, nor enhanced the protective properties of the immune globulins. Quantitative studies using graded doses of immune globulins given to infant cotton rats prior to RSV challenge showed that serum neutralizing antibody titers in excess of 1:350 conferred complete resistance to pulmonary infection, and provided the rationale for the target titer to be achieved in human clinical trials. The cotton rat model correctly predicted not only that RSV immune globulin could prevent serious pulmonary disease in high-risk infants, but also the levels of serum antibody required to achieve protection.

Equine arteritis virus

Equine arteritis virus (EAV) is a small, enveloped, positive-stranded RNA virus, in the family Arteriviridae , W.H.ich can infect both horses and donkeys. While the majority of EAV infections are asymptomatic, acutely infected animals may develop a wide range of clinical signs, including pyrexia, limb and ventral edema, depression, rhinitis, and conjunctivitis. The virus may cause abortion and has caused mortality in neonates. After natural EAV infection, most horses develop a solid, long-term immunity to the disease. Marzz and geldings eliminate the virus within 60 days, but 30 to 60% of acutely infected stallions will become persistently infected. These persistently infected animals maintain EAV within the reproductive tract, shed virus continuously in the semen, and can transmit the virus venereally. Mares infected venereally may not have clinical signs, but they shed large amounts of virus in nasopharyngeal secretions and in urine, which may result in lateral spread of the infection by an aerosol route. The consequences of venereally acquired infection are minimal, with no known effects on conception rate, but mares infected at a late stages of gestation may abort. Identification of carrier stallions is crucial to control the dissemination of EAV. The stallions can be identified by serological screening using a virus neutralization (VN) test. If positive at a titer of ≥ 1:4, the stallion should be tested for persistent infection by virus isolation from the sperm-rich fraction of the ejaculate, or by test mating Shedding stallions should not be used for breeding, or should be bred only to mares seropositive from a natural infection or from vaccination, the mares should be subsequently isolated from seronegative horses for three weeks after natural or artificial insemination. A live attenuated (ARVAC) and a formalin-inactivated (ARTERVAC) vaccine are available. Both vaccines induce virus-neutralizing antibodies, the presence of which correlates with protection from disease, abortion, and the development of a persistent infection. Serological investigations indicate that EAV has a worldwide distribution and that its prevalence is increasing. As a consequence, an increasing number of equine viral arteritis (EVA) outbreaks is being reported. This trend is likely to continue unless action is taken to slow or halt the transmission of this agent through semen.

Quantitative Reverse Transcriptase Polymerase Chain Reaction (RT-PCR) Assessment of T Helper Cell Response in a Murine Model of Respiratory Syncytial Virus (RSV) Infection • 739

Selective activation of T helper cell (Th) subsets affects disease severity in RSV infection. Animal studies have shown by Northern blot analysis that Th type 2 response to formalin-inactivated RSV vaccine is responsible for enhanced pathology in RSV disease. We developed a quantitative RT-PCR to characterize the cytokine response following primary and secondary RSV infection. Methods: Retired breeder BALB/c mice were intranasally inoculated with 106.5 plaque-forming units of RSV A2 on days 0 and +28. At various times after secondary exposure, RNA was extracted from the left lung of 2 animals and cytokine levels determined by RT-PCR. Lung RNA was reverse transcribed to complimentary DNA (cDNA) using oligo-dT primers and PCR amplified with cytokine-specific primers in the presence of increasing concentrations of primer-specific competitive fragment (CF). cDNA levels were determined from the concentration of CF at the point of equivalence of cDNA and CF on ethidium bromide stained agarose gel. Results from each time point were averaged. All samples were normalized to β-actin prior to cytokine quantitation. Results: γ-IFN was detected after primary and secondary infection, the amount increasing with time after secondary exposure. No or trace amounts of IL-2 or IL-4 was detected by this sensitive technique. Conclusion: Using this model, cytokine response to RSV infection and reinfection was quantified. As expected, primary and secondary RSV infection were associated with Th type 1 responses. Our model may be used to assess vaccines, antiviral and antiinflammatory agents for the treatment and prevention of RSV disease. Table

Venezuelan equine encephalitis virus vaccines induce mucosal IgA responses and protection from airborne infection in BALB/c, but not C3H/HeN mice

Immunization with either a live-attenuated (TC-83) or formalin-inactivated (C-84) vaccine for Venezuelan equine encephalitis (VEE) virus protected BALB/c mice from lethal VEE infection acquired subcutaneously or by aerosol. While vaccinated C3H/HeN mice were also protected from parenteral infection, neither vaccine protected these mice from an aerosol infection. The apparent vaccine failures in C3H/HeN mice could not be attributed to deficiencies in virus-neutralizing antibodies in serum, as these responses were typically of equal or higher titer than those observed in protected BALB/c mice before challenge. IgG subclass analysis offered no facile explanation: profiles of IgG2a dominance were observed in C3H/HeN mice given either vaccine and in BALB/c mice given the live-attenuated vaccine, whereas BALB/c antibody responses shifted toward IgG1 dominance after immunization with the killed C-84 vaccine. Data from immunized congenic showed that the H-2 genes from the C3H/He mice were not singularly responsible for the inability of these mice to resist aerosol infection with VEE virus. VEE virus-specific IgA responses were detected more frequently in respiratory and vaginal secretions obtained from the protected BALB/c mice.

Assault against respiratory syncytial virus: beginning of an era

On the heels of the extraordinary success of effective immunisation against Haemophilus influenzae B, the race for prevention has moved on to another virulent pathogen, respiratory syncytial virus (RSV). Early attempts to conquer RSV with a formalin-inactivated vaccine brought disaster in the form of enhanced infection on exposure to wild virus. Now, more than 25 years later, the first effective prophylactic strategy against RSV has arrived: passive immunisation with a high-titre intravenous immune globulin product, RSV-IGIV.

Generation of atypical pulmonary inflammatory responses in BALB/c mice after immunization with the native attachment (G) glycoprotein of respiratory syncytial virus.

The feasibility of using the highly purified native attachment (G) protein in a subunit vaccine against respiratory syncytial virus (RSV) was examined in a murine model with or without the fusion (F) protein of RSV and the adjuvant QS-21. The studies established that QS-21 was more potent than AIOH as an adjuvant for both F and G glycoproteins. Augmented antigen-dependent killer cell activity and complement-assisted serum neutralizing and anti-F and G protein immunoglobulin G2a antibody titers were observed. Immunization with G/QS-21 generated immune responses that were characterized by low levels of antigen-dependent killer cell activity, elevated levels of interleukin-5 (IL-5) and percentages of eosinophils in the bronchoalveolar lavage fluids after challenge, and splenic immunocytes that secreted IL-5 but not gamma interferon (IFN-gamma) after in vitro stimulation with purified whole virus antigens. The pulmonary eosinophilia was similar to that induced by a facsimile of a formalin-inactivated vaccine used in previous clinical trials and was prevented by prior in vivo treatment with anti-IL-5 but not with control immunoglobulin G or anti-IFN-gamma neutralizing monoclonal antibodies. Thus the data implied that vaccination with G/QS-21 generated helper T-cell immune responses that were type 2 in nature. Alternatively, the data suggested that the helper T-cell immune responses elicited by F/QS-21 were more type 1 in character. Neither eosinophilia nor elevated levels of IL-5 were observed in the lungs of mice after challenge. Noteworthy levels of antigen-dependent killer cell activity was observed, and splenic immunocytes secreted copious quantities of IFN-gamma. Immunization with a combination vaccine composed of highly purified native F and G proteins plus QS-21 (F+G/QS-21) resulted in augmented complement-assisted serum neutralizing antibody titers compared with vaccination with either F/QS-21 or G/QS-21 alone. However, following vaccination with F+G/QS-21, the bronchoalveolar lavage fluids contained significant increases in IL-5 and percentages of eosinophils after challenge, the spleen cells appeared to secrete less IFN-gamma after in vitro stimulation, and there was no evidence of increased numbers of antigen-dependent killer cell precursors. Taken together, the data imply that native G protein influences the nature of the immune responses elicited by F/QS-21. The results therefore suggest that G, not F, protein has more potential to bias the host for atypical pulmonary inflammatory responses.

Hepatitis A

Hepatitis A is a disease with worldwide distribution. Conditions of sanitation and hygiene determine the overall epidemiological situation in any area, but risk behaviour determines an individual's chance of exposure. The recent licensing of formalin-inactivated vaccines for the prevention of hepatitis A requires an understanding of the epidemiology in order to devise how best to use this the vaccine in individuals or populations. Although higher-risk groups can be identified in developed countries, and vaccine will be useful to those groups, it is not likely that the vaccine will have a large effect on the prevalence of hepatitis A until it becomes a part of the routine childhood immunization schedule.

Long-term duration of detectable neutralizing antibodies after administration of live-attenuated VEE vaccine and following booster vaccination with inactivated VEE vaccine

The US Army successfully developed a live-attenuated Venezuelan Equine Encephalitis (VEE) vaccina, TC-83, in 1961, and subsequently developed a formalin-inactivated vaccine, C-84, in 1974. Initial evaluation of both vaccines was promising, but no long-term safety and immunogenicity data have been reported. This study is the first analysis of the long-term safety and immunogenicity of TC-83 and C-84. From January 1976 to December 1990, 821 laboratory workers at the USAMRIID were vaccinated with a single 0.5 ml subcutaneous (s.c.) dose of TC-83; 128 were boosted with a single 0.5 ml s.c. dose of C-84. Eighty-two per cent of vaccinees responded to TC-83 with an 80% plaque reduction neutralization titer (PRNT 80) of ≥1:20. Minor side-effects were noted in 23% of vaccinees. No long-term sequelae were recorded. Kaplan-Meier analysis showed a 60% probability of vaccinees maintaining a PRNT 80 of ≥1:20 for 5.5–8 years. C-84 was given to two groups: 76 initial nonresponders to TC-83, Group A, and 52 initial responders to TC-83 whose PRNT 80 became <1:20 over time, Group B. C-84 successfully boosted 76% of Group A and 100% of Group B to a PRNT 80 ≥1:20. Kaplan-Meier analysis showed 100% probability of Group B members maintaining a titer of ≥2:20 for the duration of follow-up, which, in some cases, exceeded 10 years; while Group A had only a 60% probability of maintaining a titer for 1–2 years. Only minor local reactions to C-84 were noted in 6.3% of vaccinees. We conclude that, although TC-83 is reactogenic, when administered as the primary vaccine and C-84 is administered as a boost, these vaccines provide good long-term immunity and are safe in humans. However, a single dose vaccine that is more immunogenic and less reactogenic is needed.

Evaluation of formalin-inactivated Clostridium difficile vaccines administered by parenteral and mucosal routes of immunization in hamsters.

Clostridium difficile produces toxins that cause inflammation, necrosis, and fluid in the intestine and is the most important cause of nosocomial antibiotic-associated diarrhea and colitis. We evaluated C. difficile antigens as vaccines to protect against systemic and intestinal disease in a hamster model of clindamycin colitis. Formalin-inactivated culture filtrates from a highly toxigenic strain were administered by mucosal routes (intranasal, intragastric, and rectal) with cholera toxin as a mucosal adjuvant. A preparation of culture filtrate and killed whole cells was also tested rectally. The toxoid was also tested parenterally (subcutaneously and intraperitoneally) and by a combination of three intranasal immunizations followed by a combined intranasal-intraperitoneal boost. Serum antibodies against toxins A and B and whole-cell antigen were measured by enzyme-linked immunosorbent assay, neutralization of cytotoxic activity, and bacterial agglutination. The two rectal immunization regimens induced low antibody responses and protected only 20% of hamsters against death and 0% against diarrhea. The intragastric regimen induced high antibody responses but low protection, 40% against death and 0% against diarrhea. Hamsters immunized by the intranasal, intraperitoneal, and subcutaneous routes were 100% protected against death and partially protected (40, 40, and 20%, respectively) against diarrhea. Among the latter groups, intraperitoneally immunized animals had the highest serum anticytotoxic activity and the highest agglutinating antibody responses. Hamsters immunized intranasally and revaccinated intraperitoneally were 100% protected against both death and diarrhea. Protection against death and diarrhea correlated with antibody responses to all antigens tested. The results indicate that optimal protection against C. difficile disease can be achieved with combined parenteral and mucosal immunization.

Enhancement of protective immune responses to Venezuelan equine encephalitis (VEE) virus with microencapsulated vaccine

Venezuelan equine encephalomyelitis (VEE) virus is a mosquito-borne arbovirus of major human health significance in the New World. Currently two forms of VEE virus are used for immunization of humans and horses, i.e. a live attenuated and a formalin-inactivated vaccine. Clinical evidence suggests that these vaccines are not fully efficacious and may produce certain undesirable side-effects. In the present study, microspheres composed of biocompatible and biodegradable poly (DL-lactide-co-glycolide) (DL-PLG) were evaluated for their effectiveness as a delivery system of whole, inactivated VEE virus vaccine for the induction of protective immune responses. Mice receiving 50 μg VEE virus in microspheres composed of an equimolar ratio of DL-lactide and glycolide (50:50 DL-PLG) exhibited a primary circulating IgG antibody response which was approximately 32-times higher than the response induced with the same dose of unencapsulated (free) virus. A similar difference in responses was seen with antigen doses ranging from 3.1 to 50 μg. A rapid increase in antibody activity was seen after the secondary immunization (day 50). Formalin fixation of inactivated VEE virus was important for immunogenicity since the circulating anti-VEE virus antibody response induced with microencapsulated nonformalin-fixed virus vaccine was lower than that induced with microencapsulated formalin-fixed virus vaccine. Furthermore, at low antigen concentrations, DL-PLG microsphere vaccines prepared with the solvent methylene chloride induced higher antibody responses than those prepared using ethyl acetate as the solvent. Microencapsulated vaccine also induced higher VEE virus neutralization titers than did free virus vaccine. Finally, the microencapsulated virus was more effective than the free virus in inducing immune responses protective against systemic challenge with virulent VEE virus. These results demonstrate that DL-PLG microspheres containing formalin-fixed, inactivated VEE virus were effective in augmenting circulating IgG antibody levels and neutralization titers to the VEE virus following systemic immunization and in affording enhanced protection against systemic challenge with virulent VEE virus. The effects of antigen form and the microsphere processing solvent on the immunogenicity of the vaccine are discussed.

Enhanced pulmonary histopathology induced by respiratory syncytial virus (RSV) challenge of formalin-inactivated RSV-immunized BALB/c mice is abrogated by depletion of interleukin-4 (IL-4) and IL-10.

In previous studies, children immunized with a formalin-inactivated respiratory syncytial virus vaccine (FI-RSV) developed severe pulmonary disease with greater frequency than did controls during subsequent natural RSV infection. In earlier efforts to develop an animal model for this phenomenon, extensive pulmonary histopathology developed in FI-RSV-immunized cotton rats and mice subsequently challenged with RSV. In mice, depletion of CD4+ T cells at the time of RSV challenge completely abrogated this histopathology. Furthermore, the predominant cytokine mRNA present in lungs of FI-RSV-immunized mice during subsequent infection with RSV was that characteristically secreted by Th2 T cells, namely interleukin-4 (IL-4). In the present studies, we sought to determine the relative contributions of gamma interferon (IFN-gamma), IL-2, IL-4, and IL-10 to the lymphocytic infiltration into the lungs observed following RSV challenge of mice previously immunized with FI-RSV. Mice previously immunized with FI-RSV or infected with RSV were depleted of IFN-gamma, IL-2, IL-4, or IL-10 immediately before RSV challenge, and the magnitude of inflammatory cell infiltration around bronchioles and pulmonary blood vessels was quantified. The phenomenon of pulmonary-histopathology potentiation by FI-RSV was reproduced in the present study, thereby allowing us to investigate the effect of cytokine depletion on the process. Simultaneous depletion of both IL-4 and IL-10 completely abrogated pulmonary histopathology in FI-RSV-immunized mice. Depletion of IL-4 alone significantly reduced bronchiolar, though not perivascular, histopathology. Depletion of IL-10 alone had no effect. Depletion of IFN-gamma, IL-2, or both together had no effect on the observed histopathology. These data indicate that FI-RSV immunization primes for a Th2-, IL-4-, and IL-10-dependent inflammatory response to subsequent RSV infection. It is possible that this process played a role in enhanced disease observed in infants and children immunized with FI-RSV.

Immunogenicity, safety and tolerability of varying doses and regimens of inactivated hepatitis A virus vaccine in Navajo children

The Navajo are known to be at high risk for hepatitis A virus (HAV) infection. This study investigated the safety and immunogenicity of an investigational, alum-adjuvanted, formalin-inactivated HAV vaccine (VAQTA) developed by Merck Research Laboratories in Navajo children. One hundred two of 212 children, ages 4 to 12 years, were HAV-seronegative (< 10 mIU/ml by an enhanced sensitivity modification of the HAVAB; Abbott). Ninety of these children received the HAV vaccine. Study participants were given vaccines containing various viral protein concentrations: Group A (n = 18), 6 units; Group B (n = 36), 13 units; and Group C (n = 36), 25 units HAV protein (1 unit approximately 1 ng viral protein antigen). Three-dose (0, 8, 24 weeks) and two-dose (0, 24 weeks) regimens were compared in subgroups within B and C. The vaccine was well-tolerated and there were no serious adverse reactions; no vaccinee developed hepatitis A. After 1 dose 82 to 100% of children seroconverted (> or = 10 mIU/ml, modified HAVAB; Abbott) and 100% seroconverted after 2 doses. After 1 dose the geometric mean titer for antibody was: Group A, 22 mIU/ml; Group B, 18 mIU/ml; and Group C, 38 mIU/ml. After 3 doses geometric mean titers increased to 10,106 mIU/ml in Group A, 7258 mIU/ml in Group B and 11,856 mIU/ml in Group C. Further field studies are indicated to evaluate its use in high risk populations, such as the Navajo.

Vaccine prophylaxis of Q fever: A follow‐up study of the efficacy of Q‐Vax (CSL) 1985‐1990

To examine the efficacy of various batches of a formalin-inactivated whole cell Coxiella burnetti vaccine (Henzerling strain, Phase 1 [Q-Vax, CSL]) in the prevention of Q fever among abattoir workers. The study was a retrospective cohort survey of all employees at three South Australian abattoirs to determine the incidence of Q fever among vaccinated and unvaccinated employees during the period 1985 to 1990. There were two cases of Q fever among 2555 vaccinated employees of the three abattoirs, compared with 55 cases among 1365 unvaccinated employees. The two Q fever cases in vaccinated employees were within a few days of vaccination, before immunity had developed, and represented a coincidence of natural infection and vaccination. Protective efficacy was 100%, even with a batch of Q-Vax containing 20 micrograms/dose rather than the standard dose of 30 micrograms/dose. Vaccination was effective for at least five years, although it was uncertain whether this was due to the vaccine per se or to a combination of vaccine immunity reinforced by periodic natural exposure.

Experiments with inactivated visna-maedi vaccine.

Sixteen Icelandic sheep were vaccinated against Visna-Maedi infection with formalin inactivated whole virus vaccine to which alun was added as an adjuvant. Western Blots showed that all the vaccinees responded to the major proteins of the virus. They all developed neutralizing antibodies ranging in titers from 1/8-1/256 at maximum for individual sheep. Passive transfer of antibodies and neutralizing activity from vaccinated ewes to their lambs was demonstrated. Exposure of the 16 vaccinees and their 16 unvaccinated control sheep to natural infection is attempted by housing them with heavily infected sheep. After 9 months of exposure there is no seroconversion in the unvaccinated group. Only time can tell, whether or not such conventional vaccination with inactivated vaccine protects the vaccinees.

A vaccinia virus — herpes simplex virus (HSV) glycoprotein B1 recombinant or an HSV vaccine overcome the HSV type 2 induced humoral immunosuppression and protect against vaginal challenge in BALB/c mice

Primary infections with herpes simplex virus type 2 (HSV-2) suppress the antibody response to secondary HSV-1 and -2 infections in the BALB/c mouse. In contrast, a challenge by the i.p. route using a vaccinia virus-HSV-1 glycoprotein B (VV gB1) recombinant induces a significant enhancement of the antibody response. This booster reaction is also observed if a challenge with a formalin-inactivated HSV-1 vaccine is performed. Although no or low humoral and vaginal antibodies are detectable after a single i.p. infection with the VV gB1 recombinant or the HSV-1 vaccine, protection against vaginal challenge with HSV-2 is induced. This points to the important role of cellular immunity for vaginal infections.

Immunoblot analyses of chimpanzee sera after infection and after immunization and challenge with Mycoplasma pneumoniae.

Consecutive weekly or biweekly serum specimens obtained during a 3- or 4-month study from 16 chimpanzees were examined by immunoblot analyses to identify the immunogenic components of Mycoplasma pneumoniae. Six experimentally infected chimpanzees showed significant signs of overt disease, including cough, pharyngitis, rhinitis, fever, and loss of appetite. The sera of these infected chimpanzees recognized from 17 to 20 protein bands. Two control chimpanzees that were not inoculated were included in the study. Three chimpanzees immunized with a formalin-inactivated OSU-1A vaccine and three chimpanzees immunized with an experimental acellular vaccine showed minimal signs of disease on challenge. After challenge, the serum immunoblot responses of the immunized chimpanzees were similar to those of the infected chimpanzees. Before challenge, the sera of two previously infected chimpanzees recognized protein bands of 169 (which comigrated with the P1 adhesin), 148, 130, 117, 86, 61, 44, 35, 30, and 29 kDa. After challenge, the previously infected chimpanzees showed the most intense serum immunoblot responses and were most protected against colonization and disease. The sera from each of the 16 chimpanzees examined recognized a large number of immunogenic components, and the serum immunoblot responses were virtually identical to those of patients. Sera from each chimpanzee and patient recognized 169-, 148-, 130-, 117-, 86-, 44-, and 35-kDa bands and many of them recognized 67-, 63-, 61-, 56-, 32-, 30-, and 29-kDa protein bands.

A formalin-inactivated vaccine protects against mucosal papillomavirus infection: a canine model.

A formalin-inactivated canine oral papilloma homogenate was used as a vaccine to prevent infection by the oncogenic, mucosotropic canine oral papillomavirus (COPV) in beagle dogs. Twenty-six dogs received 2 doses of phosphate-buffered saline intradermally and 99 dogs received 2 doses of the inactivated vaccine. One month after the second dose all dogs were challenged with infectious COPV by scarification of the oral mucosa. All of the control dogs developed papillomas by 6-8 weeks after challenge while none of the vaccinated dogs did. This vaccine has been used successfully in approximately 60,000 line bred beagles with no untoward effects and with long-lasting protection. These data demonstrate that a systemically administered, formalin-inactivated vaccine can protect against mucosal infection by COPV and suggest approaches for the development of human papillomavirus vaccines.

Prospects for a respiratory syncytial virus vaccine

There is an urgent need to develop a safe and efficacious vaccine capable of protecting infants and young children against the severe respiratory tract infections caused by Respiratory Syncytial Virus (RSV). Neither formalin-inactivated nor live-attenuated RSV vaccines provided adequate protection in clinical trials. Moreover, the formalin-inactivated vaccine caused exacerbated disease in some vaccinees following exposure to wild-type virus. The mechanism of disease potentiation has not been defined although both viral and non-viral components have been implicated. Alternate vaccine strategies currently under consideration include the production of : i) purified viruses inactivated by procedures other than formalin ; ii) attenuated viruses which are both non-virulent and genetically stable ; iii) native or recombinant subunit preparations including both the F and G surface glycoproteins ; iv) live recombinant viruses expressing the pertinent viral genes ; and v) synthetic peptides corresponding to selected B- and T-cell epitopes. While promising results have been obtained with some of these approaches, it is not yet possible to select one strategy which yields a vaccine preparation suitable for clinical evaluation in seronegative infants. This will necessitate the continued pursual of a multi-faceted approach to RSV vaccine development. Un vaccin efficace et bien toléré contre les infections à VRS paraît une nécessité urgente pour protéger les nourrissons et les très jeunes enfants. Ni les vaccine VRS inactivés par le formol, ni les vaccins vivants atténués n'ont apporté une protection correcte au cours des essais cliniques. Puis encore, le vaccin formolé provoquait une maladie aggravée chez certains vaccinés après une exposition au virus sauvage. Le mécanisme de la potentialisation de la maladie n'a pas été élucidé bien que les composants viraux et non viraux aient été impliqués conjointement. Les autres voies de recherche vaccinale actuellement retenues, comportent la production : 1) des virus purifiés inactivés par d'autres techniques que le formol ; 2) des virus atténués à la fois non virulents et stables génétiquement ; 3) des sous-unités naturelles ou recombinantes incluant les glycoprotéines de surface F et G ; 4) des virus recombinants vivants exprimant les gènes viraux essentiels ; 5) des peptides synthétiques correspondant aux épitopes sélectionnés des cellules B et T. Bien que des résultats prometteurs aient été obtenus pour certaines de ces stratégies il n'est pas encore possible d'en choisir une qui offre une préparation vaccinale acceptable pour une évaluation/clinique chez le nourrisson séronégatif. Ceci nécessitera de poursuivre cette approche multifactorielle des vaccins VRS.

The molecular biology of tick-borne encephalitis virus. Review article.

Tick-borne encephalitis (TBE) virus is a member of the flavivirus genus and the family Flaviviridae. Like other flaviviruses such as yellow fever, Japanese encephalitis or the dengue viruses, it is an important human pathogen, endemic in many European countries, Russia and China. The disease can be effectively prevented by vaccination with a formalin-inactivated whole virus vaccine. In recent years major advances have been made in the understanding of the molecular biology of TBE virus, including the complete sequence analysis of the genomic RNA of the European and Far Eastern strains. As shown in these studies, the virion RNA contains a single long open reading frame that codes for the structural proteins at the 5' end and the nonstructural proteins at the 3' end. Co- and posttranslational cleavages by a viral and cellular proteases lead to the formation of individual viral proteins. The mature virion is composed of an isometric capsid surrounded by a lipid envelope with two membrane-associated proteins. One of these, protein E, is of paramount importance for several important viral functions, especially during the entry phase of the viral life cycle. Protein E is also responsible for the induction of a protective immune response. A detailed map of antigenic sites has been established and the structure of an anchor-free form of E is currently being investigated by X-ray diffraction analysis. Understanding the molecular basis of the functions of this protein together with the knowledge of its three-dimensional structure may provide clues for developing specific antiviral agents. Protein E has also been shown to be an important determinant of virulence, with single amino acid substitutions at selected sites leading to attenuation. Engineering of such mutations into cDNA clones to produce new recombinant viruses may open up new avenues for the development of live vaccines.