Helma W. Vos

Safety of modified vaccinia virus Ankara (MVA) in immune-suppressed macaques

Modified vaccinia virus Ankara (MVA)-based recombinant viruses have been shown to be potent vaccine candidates for several infectious and neoplastic diseases. Since a major application of these live, replication-deficient vectors would be their use in immunocompromised or potentially immunocompromised individuals, a preclinical safety study was carried out. Macaques were inoculated with high doses of MVA (10(9)) via various routes, after immune-suppression by total-body irradiation, anti-thymocyte globulin treatment, or measles virus (MV) infection. No clinical, haematological or pathological abnormalities related to MVA inoculation were observed during a 13-day follow-up period. The presence of MVA genomes was demonstrated by nested PCR during the course of the experiment in all macaques, but from none of these animals replication competent MVA could be reisolated. These data suggest that MVA can safely be used as a basis for recombinant human vaccines, and that it is also safe for use in immunocompromised individuals.

Protective Immunity in Macaques Vaccinated with a Modified Vaccinia Virus Ankara-Based Measles Virus Vaccine in the Presence of Passively Acquired Antibodies

ABSTRACT Recombinant modified vaccinia virus Ankara (MVA), encoding the measles virus (MV) fusion (F) and hemagglutinin (H) (MVA-FH) glycoproteins, was evaluated in an MV vaccination-challenge model with macaques. Animals were vaccinated twice in the absence or presence of passively transferred MV-neutralizing macaque antibodies and challenged 1 year later intratracheally with wild-type MV. After the second vaccination with MVA-FH, all the animals developed MV-neutralizing antibodies and MV-specific T-cell responses. Although MVA-FH was slightly less effective in inducing MV-neutralizing antibodies in the absence of passively transferred antibodies than the currently used live attenuated vaccine, it proved to be more effective in the presence of such antibodies. All vaccinated animals were effectively protected from the challenge infection. These data suggest that MVA-FH should be further tested as an alternative to the current vaccine for infants with maternally acquired MV-neutralizing antibodies and for adults with waning vaccine-induced immunity.

Immunization of macaques with formalin-inactivated respiratory syncytial virus (RSV) induces interleukin-13-associated hypersensitivity to subsequent RSV infection

ABSTRACT Respiratory syncytial virus (RSV) is a major cause of severe respiratory disease in infants and the elderly. RSV vaccine development has been hampered by results of clinical trials in the 1960s, when formalin-inactivated whole-RSV preparations adjuvated with alum (FI-RSV) were found to predispose infants for enhanced disease following subsequent natural RSV infection. We have reproduced this apparently immunopathological phenomenon in infant cynomolgus macaques and identified immunological and pathological correlates. Vaccination with FI-RSV induced specific virus-neutralizing antibody responses accompanied by strong lymphoproliferative responses. The vaccine-induced RSV-specific T cells predominantly produced the Th2 cytokines interleukin-13 (IL-13) and IL-5. Intratracheal challenge with a macaque-adapted wild-type RSV 3 months after the third vaccination elicited a hypersensitivity response associated with lung eosinophilia. The challenge resulted in a rapid boosting of IL-13-producing T cells in the FI-RSV-vaccinated animals but not in the FI-measles virus-vaccinated control animals. Two out of seven FI-RSV-vaccinated animals died 12 days after RSV challenge with pulmonary hyperinflation. Surprisingly, the lungs of these two animals did not show overt inflammatory lesions. However, upon vaccination the animals had shown the strongest lymphoproliferative responses associated with the most pronounced Th2 phenotype within their group. We hypothesize that an IL-13-associated asthma-like mechanism resulted in airway hyperreactivity in these animals. This nonhuman primate model will be an important tool to assess the safety of nonreplicating candidate RSV vaccines.

Impaired cellular immune response in harbour seals (Phoca vitulina) feeding on environmentally contaminated herring.

In a 2.5‐year immunotoxicological study, two groups of captive harbour seals (Phoca vitulina) were fed herring from the heavily polluted Baltic Sea or from the relatively uncontaminated Atlantic Ocean. Blood samples were collected at regular intervals, and functional immunological parameters were monitored. T cell mitogen and mixed lymphocyte‐induced proliferative responses of peripheral blood mononuclear cells (PBMC) obtained from seals fed Baltic herring were significantly reduced over the course of the experiment. Upon immunization with rabies virus antigen (RV) and tetanus toxoid (TT), specific proliferative responses of PBMC from the seals fed Baltic herring were also significantly reduced. Impairment of T cell‐mediated immune responses became especially apparent during the second year on the respective diets, and correlated significantly to 2,3,7,8‐tetrachloro‐dibenzo‐p‐dioxin toxic equivalent levels in blubber biopsies taken from the seals after 2 years on the respective diets. Humoral immune responses, including lipopolysaccharide (LPS)‐induced lymphoproliferative responses, in vitro immunoglobulin production by PBMC, as well as RV‐, TT‐ and poliovirus‐specific serum antibody responses following immunization, remained largely unaffected. We conclude that suppression of the cellular immune response in the seals fed Baltic herring was induced by the chronic exposure to immunotoxic environmental contaminants accumulated through the food chain. Since cellular immune responses are known to be of crucial importance in the clearance of morbillivirus infections, these results suggest that environmental pollution‐related immunosuppression may have contributed to the severity and extent of recent morbillivirus‐related mass mortalities among marine mammals.

Characterization of morbilliviruses isolated from dolphins and porpoises in Europe

A previously unidentified morbillivirus was isolated from two harbour porpoises (Phocoena phocoena) that had died in the Dutch Waddensea (North Sea) in 1990. This porpoise morbillivirus (PMV) and a dolphin morbillivirus (DMV), which had recently caused a heavy mortality in Mediterranean striped dolphins (Stenella coeruleoalba), were compared antigenically with other members of the genus Morbillivirus, including the newly recognized phocine distemper virus type 1. DMV and PMV proved to be similar but distinct morbillivurses, closely related to rinderpest virus and peste-des-petitsruminants virus. Cell cultures of cetacean, pinniped, ruminant and canine origin showed a different pattern of susceptibility to DMV and PMV infection. Ruminants and dogs proved to be susceptible to experimental infection with DMV and PMV, which both caused a transient leukopenia most pronounced in the ruminants. Pre-exposure of dogs to DMV and PMV protected them from developing CDV viraemia and clinical signs upon challenge infection with virulent CDV. A serological survey among stranded animals of different cetacean species in Europe indicated that infections with DMV- and PMV-like morbilliviruses are not uncommon among these aquatic mammals.

Morbillivirus infections of aquatic mammals: newly identified members of the genus

Several disease outbreaks, which have caused the deaths of many thousands of seals and dolphins during the last decade, have now been attributed to infections with newly identified Morbilliviruses. Outbreaks in the late eighties amongst harbour seals (Phoca vitulina) and grey seals (Halichoerus grypus) in northwestern Europe and amongst baikal seals (Phoca sibirica) in Siberia were caused by the newly discovered phocine distemper virus and by a strain of canine distemper virus, respectively. Although closely related these two viruses were not identical. They were more distantly related to the viruses which caused mass mortality amongst striped dolphins (Stenella coeruleoalba) in the Mediterranean sea in the early nineties. This dolphin morbillivirus was shown to be closely related to a virus that was found in harbour porpoises (Phocoena phocoena) which had stranded at the coasts of northwestern Europe in the late eighties: porpoise morbillivirus. The present knowledge of the genetic and antigenic relationships of these apparently new members of the genus Morbillivirus with the established members of the genus is presented. In addition, the origin and epizootiological aspects of these newly discovered viruses are discussed. Finally experimental evidence that environmental pollution may have contributed to the severity and extent of these infections in recent years is presented.

Induction of protective immunity to Theileria annulata using two major merozoite surface antigens presented by different delivery systems

Allelic forms (Tams1-1 and Tams1-2) of the major merozoite surface antigen gene of Theileria annulata have recently been expressed in Escherichia coli and in Salmonella typhimurium aroA vaccine strain SL3261. To test the potential of subunit vaccines against T. annulata infection, we immunized four groups of three calves with either recombinant (re-) (Tams1-1 and Tams1-2) proteins or naked DNA encoding these antigens. Group I was immunized intramuscularly with both re-proteins incorporated into immunostimulating complexes (ISCOMs). Group II was inoculated intramuscularly with naked plasmid DNA encoding Tams1-1 and Tams1-2. Groups III and IV received S. typhimurium SL3261 [pSTams1-1][pIP5] and SL3261 [pSTams1-2] [pIP5] subcutaneously and orally, respectively. A final group of three animals (Group V) served as an unimmunized control group. Four weeks after the last immunization all calves were challenged with a T. annulata stabilate generated from blood of an infected animal with 30% piroplasm parasitaemia. All calves vaccinated with ISCOMs proved to be protected from T. annulata infection and had generated antibodies against both re-(Tams1-1 and Tams1-2) at the time of challenge. In two of these animals the antibody had a surface binding profile by IFAT. Two of three calves immunized with naked DNA also proved to be protected, but none of the animals had generated any detectable antibodies against the recombinants. Salmonella-based delivery of the recombinants did not induce any protection; two of six animals died of theileriosis and there was no difference between subcutaneous or oral administration. These preliminary results show that re-(Tams1-1 and/or Tams1-2) may elicit protective immune responses in cattle, depending on the antigen delivery system.

Characterization of phocid herpesvirus-1 and -2 as putative alpha- and gammaherpesviruses of North American and European pinnipeds

To study the relationships between herpesvirus recently isolated from different pinniped species, antigenic and genetic analyses were performed. First, herpesviruses isolated from North American harbour seals (Phoca vitulina), a Californian sea lion (Zalophus californianus) and a European grey seal (Halichoerus grypus) were examined in an enzyme immunoassay (EIA) with a panel of monoclonal antibodies which had previously been shown to allow typing of herpesviruses from European harbour seals into two distinct virus types: phocid herpesvirus type-1 and type-2 (PhHV-1 and PhHV-2). The EIA data showed that all but one of the isolates from seals ranging in United States coastal waters were PhHV-2-like while the European grey seal herpesvirus was PhHV-1-like. Genetic characterization was facilitated by PCR analysis using primers based on conserved regions of the glycoprotein B and D (gB and gD) genes of the antigenically closely related canid (CHV) and felid (FHV) herpesvirus. Specific amplified products were obtained with five isolates antigenically characterized as PhHV-1-like but not with five PhHV-2-like isolates. Sequence analysis of the PCR products confirmed greatest similarity to members of the genus Varicellovirus of the Alphaherpesvirinae and in particular to CHV. Sequence analysis of two EcoRI fragments of the PhHV-2 genome (European isolate 7848) revealed greatest similarity to gammaherpesviruses and in particular equine herpesvirus-2. Although an unambiguous subgrouping was not feasible, this is the first evidence that PhHV-2 may be a putative gammaherpesvirus of pinnipeds.

Measles virus fusion protein- and hemagglutinin-transfected cell lines are a sensitive tool for the detection of specific antibodies by a FACS- measured immunofluorescence assay

A FACS-measured immunofluorescence assay was developed for the detection of antibodies directed against the hemagglutinin (H) and fusion (F) glycoproteins of measles virus (MV). Human melanoma cell lines transfected with either the MV H or F genes, which showed a high surface expression of the respective proteins in their native conformation, were used as target cells. The cells were incubated with diluted plasma samples, and stained subsequently with FITC-conjugated secondary antibodies. The FACS-measured fluorescence signals correlated directly with the amount of specific immunoglobulins over a wide concentration range. The use of different conjugates enabled the separate detection of MV-specific IgG, IgM, IgA and IgG subclasses, with relatively low backgrounds. Hemagglutinin-specific IgG, IgM and IgA fluorescence signals were shown to correlate well with MV-specific IgG ELISA titers and MV-specific IgM or IgA capture ELISA OD450-values, respectively. The polyclonal conjugates with specificity for human immunoglobulins offered sufficient cross-reactivity to detect MV-specific IgG, IgM and IgA in plasma samples of cynomolgus macaques, making this technique a useful tool for studying serological responses in vaccination and challenge experiments in non-human primate models.

SEROLOGIC SURVEY FOR PHOCID HERPESVIRUS-1 AND -2 IN MARINE MAMMALS FROM ALASKA AND RUSSIA

Blood samples were collected from 1,042 marine mammals off the coast of Alaska (USA) and Russia during the period 1978 to 1994. Eight species of pinnipeds were represented. Sera were tested for presence of neutralizing antibodies to both the PB84 isolate of phocid herpesvirus-1 (PhHV-1) and the 7848/Han90 strain of phocid herpesvirus-2 (PhHV-2). Species-specific antibody prevalences ranged from 22% to 77% for PhHV-1 and 11% to 50% for PhHV-2. Species-specific antibody prevalences for PhHV-1 were greater than or equal to prevalences for PhHV-2. For both viruses and each host species, differences in antibody prevalences were not related to: (1) sex, (2) location of capture, or (3) year of collection. Antibody prevalence of PhHV-1 in walruses (Odobenus rosmarus) could be quantitatively predicted as a function of age. These two viruses have distinct biological properties and based on current data the epizootiology of the two viruses is different, as well. No evidence of herpesvirus-induced mortality has been detected in areas included in this survey. Based on results of this survey, neither PhHV-1 nor PhHV-2 are considered significant mortality factors in mammals which inhabit the marine environment off the coast of Alaska or Russia.