Protective immunity by an engineered DNA vaccine for Mayaro virus.

Mayaro virus (MAYV) of the genus alphavirus is a mosquito-transmitted emerging infectious disease that causes an acute febrile illness, rash, headaches, and nausea that may turn into incapacitating, persistent arthralgias in some victims. Since its discovery in Trinidad in 1954, cases of MAYV infection have largely been confined there and to the northern countries of South America, but recently, MAYV cases have been reported in some island nations in the Caribbean Sea. Accompanying these reports is evidence that new vectors, including Aedes spp. mosquitos, recently implicated in the global spread of Zika and chikungunya viruses, are competent for MAYV transmission, which, if true, could facilitate the spread of MAYV beyond its current range. Despite its status as an emerging virus, there are no licensed vaccines to prevent MAYV infection nor therapeutics to treat it. Here, we describe the development and testing of a novel DNA vaccine, scMAYV-E, that encodes a synthetically-designed consensus MAYV envelope sequence. In vivo electroporation-enhanced immunization of mice with this vaccine induced potent humoral responses including neutralizing antibodies as well as robust T-cell responses to multiple epitopes in the MAYV envelope. Importantly, these scMAYV-E-induced immune responses protected susceptible mice from morbidity and mortality following a MAYV challenge.

Efficacy and safety of an inactivated vaccine against Salmonid alphavirus (family Togaviridae).

Pancreas disease (PD) in salmonid fish is caused by an infection with Salmonid alphavirus (SAV) and remains as one of the major health problems in the European fish farming industry. Sequence studies have revealed a genetic diversity among viral strains. A subtype of SAV (SAV3) is causing an epizootic in farmed salmonids in Norway. Here we evaluate efficacy and safety of an inactivated virus vaccine based on ALV405, a strain of SAV3 that was isolated from Norwegian salmon. The vaccine provided an average relative percent survival (RPS) of 98.5 in an intraperitoneal challenge model, and induced nearly total protection against PD in a cohabitant challenge model. It provided significant protection against SAV-induced mortality also in a field trial under industrial conditions. Local reactions seen as melanization and adhesions in the visceral cavity were less severe than those induced by two commercial vaccines. Finally, we demonstrated that the protection is not impaired when the ALV405 antigen is combined with other viral or bacterial antigens in a polyvalent vaccine. The results confirm that efficient and safe protection against SAV infection and development of PD is possible using an inactivated virus vaccine, both alone and as a component in a polyvalent vaccine.

A delayed-type hypersensitivity-inducing T-cell epitope of Semliki Forest virus mediates effective T-helper activity for antibody production.

The rational development of peptide vaccines requires the identification of both B- and T-cell epitopes. In this study, potential T-helper cell epitopes of Semliki Forest virus (SFV) were identified on the basis of their ability to induce delayed-type hypersensitivity (DTH) in mice using recombinant SFV fragments produced as hybrid proteins with beta-galactosidase in Escherichia coli and synthetic peptides coupled to beta-galactosidase. Although the tested fragments spanned almost the entire amino acid sequence of the structural proteins of SFV, only one DTH-inducing region (located between amino acid 137 and 151 of the SFV E2 membrane protein) was identified. Peptides containing this E2 region stimulated lymph node cells from SFV-primed mice in vitro. The ability of the identified T-cell epitope to induce a specific T-helper response in mice was evaluated using synthetic peptides that contained combinations of the DTH-inducing region and different previously identified linear B-cell epitopes of E2. These peptides proved able to induce an antipeptide IgG response in mice in an H-2d-restricted fashion. One of the peptides was also able to induce high titres of IgG reactive with SFV-infected cells and protected 70-100% of the peptide-immunized mice after challenge with virulent SFV. Our findings suggest that DTH and T-helper activity are mediated by different doses of the same T-cell epitope.

A vaccine against Semliki Forest virus consisting of a monoclonal anti-idiotypic antibody cross-linked to a protein which contains virus-specific T-helper cell epitopes.

A recombinantly expressed protein, consisting of cro-beta-galactosidase at the N-terminus and amino acid residues 115 to 151 of the E2 membrane of Semliki Forest virus (SFV) at the C-terminus containing two T-helper cell epitopes of SFV, was cross-linked with glutaraldehyde to a noninternal image monoclonal anti-idiotypic antibody (ab2 alpha MAb) able to induce SFV-neutralizing anti-anti-idiotypic (ab3) antibodies in BALB/c mice. This vaccine, which might potentially induce SFV-specific T-helper cell memory, established in BALB/c mice a state of protective immunity against virulent SFV within 10 days of immunization. A steady rise in serum neutralization titre occurred from day 7 to day 28 after primary anti-idiotypic immunization, levelling off thereafter. In primarily immunized mice significant rises of serum neutralization titres, which could be indicative for an operational T-helper cell memory, were not observed after challenge on day 35 with virulent SFV. The results suggest that SFV is neutralized by ab3 antibodies shortly after challenge, preventing, thereby, virus multiplication to levels sufficient to provoke a measurable booster response.

Pestivirus infection of ruminants in Australia.

Pestivirus infections are commonly diagnosed in cattle but are relatively uncommon in other ruminant species in Australia. Virus isolation is a very reliable technique for detecting pestivirus in specimens, especially when group reactive monoclonal antibodies are used with immunoperoxidase staining to detect non-cytopathogenic virus. Care must be taken to prevent adventitious pestivirus contamination of serum or cells used for cell culture. A recently developed antigen capture enzyme-linked immunosorbent assay has been extensively evaluated and found to be extremely accurate. This test is also much quicker and less expensive than virus isolation. Procedures are outlined to reliably certify animals to be free of pestivirus infection for export or as donors of semen or embryos.

Immunogenicity and vaccine efficacy of synthetic peptides containing Semliki Forest virus B and T cell epitopes.

A synthetic peptide that contains a Semliki Forest virus (SFV) B cell epitope, located at amino acid positions 240 to 255 of the E2 protein, and an SFV T helper (Th) cell epitope, located at positions 137 to 151 of the E2 protein, evoked high titres of SFV-reactive antibodies in H-2d mice. Although the peptide-induced antibodies did not neutralize SFV in vitro, 70 to 100% of the peptide-immunized mice were protected against SFV, even when viral challenge was presented 4 months after immunization. The protection could be transferred by anti-peptide serum, indicating that antibodies were responsible for the protection. When the Th cell epitope of this protective peptide was replaced by an influenza virus Th cell epitope or by another SFV Th cell epitope, the resulting peptides induced lower non-neutralizing SFV-reactive antibody titres and protected a correspondingly lower percentage of mice (50% and 30%, respectively). A peptide with the same Th cell epitope as the best protective peptide but with a less effective SFV B cell epitope protected only 33% of the mice. These results indicate that protection against SFV by a synthetic peptide is primarily dependent on its ability to induce adequate amounts of antibodies with relevant specificity and sufficient affinity; the ability to induce a relevant (SFV-specific) T memory response played only a minor role in protection.

Characterization of a major neutralization domain of Ross river virus using anti-viral and anti-peptide antibodies.

The E2 glycoprotein of the alphavirus Ross River virus (RRV) contains three defined neutralization epitopes (a, b1 and b2) with determinants located between amino acids 216 and 251 in the linear sequence (Vrati et al., 1988, Virology 162, 346-353). The antigenic structure of this region has been examined using hyperimmune mouse antiserum against RRV and antiserum against four synthetic peptides representing linear amino acid sequences in the neutralization region of E2. In plaque reduction neutralization tests using hyperimmune antiserum to RRV, an RRV mutant altered at all three neutralization epitopes was markedly more resistant than the parental virus; variants altered at single epitopes could not be distinguished in these tests. Sera from mice immunized with synthetic RRV E2 peptides conjugated to keyhole limpet haemocyanin reacted, in a direct ELISA, with the specific region of RRV represented by the peptide. The same sera did not neutralize or immunoprecipitate RRV in solution or bind to RRV in a capture ELISA. The RRV peptides did not prime mice to react to a subimmunogenic dose of RRV; they did not bind monoclonal or polyclonal antibodies to RRV. We conclude that a significant proportion of the neutralizing antibody response in mice is elicited by epitopes a, b1, and b2 of RRV E2 and that the sites to which neutralizing antibodies bind are formed by complex folding.

Studies on linear epitopes of Semliki Forest virus in protection studies against lethal challenge virus infection.

Purified reduced and non-reduced glycoproteins E1 and E2 of Semliki Forest Virus (SFV) were used to investigate the protection potency to prevent clinical disease after lethal virus challenge. In parallel synthetic oligopeptides deduced from conserved regions of the nucleotide sequences coding for the glycoproteins E1 and E2 were included. It could be demonstrated that both reduced and non-reduced glycoprotein preparations induced protection against lethal virus challenge, whereas the oligopeptides did not. The role of linear epitopes in immunity and their potential use as synthetic vaccines against Alphaviruses are critically discussed.

Delineation of protective epitopes on the E2-envelope glycoprotein of Semliki Forest virus.

Two short linear peptides, 17 and 14 amino acids long, on the Semliki Forest virus (SFV) E2-envelope polypeptide are shown to be involved in the protection of mice against lethal challenge with SFV. Peptides corresponding to these two regions, designated H and L, were selected for study on the basis of our model for prediction of protective epitopes on E2 polypeptide of alphaviruses. These peptides were produced in Escherichia coli as recombinant proteins fused to the amino terminus of beta-galactosidase. Both the H epitope (amino acid positions 227-243 on E2) and L epitope (amino acid positions 297-310) are recognized by antibodies raised against SFV, and both trigger antibodies that interact with native SFV-E2. Vaccination of mice with the H-beta-galactosidase polypeptide confers 64-87% protection against a lethal viral challenge (250 LD50), and immunization with L-beta-galactosidase leads to 23-66% protection of challenged mice. The efficacy of the L-based synthetic vaccine could be improved further (up to 100% protection) by presentation of this epitope as a dimer fused to beta-galactosidase. These results provide evidence that the algorithm and the methodology proposed by us previously are effective tools for identification of linear protective epitopes on E2-envelope of SFV.

Identification of linear epitopes on Semliki Forest virus E2 membrane protein and their effectiveness as a synthetic peptide vaccine.

Semliki Forest virus (SFV) infection of mice was used as a model to study the applicability of synthetic peptides containing only linear epitopes as viral vaccines. The identification of linear epitopes with vaccine potential on the E2 membrane protein of SFV was based on the binding of SFV-specific antibodies to a set of overlapping synthetic hexapeptides (Pepscan) representing the whole E2 amino acid sequence. The 14 available E2-specific monoclonal antibodies which were protective in vivo proved to be unsuitable for the identification of linear epitopes because they recognized only conformational epitopes, as indicated by their lack of reactivity with unfolded, reduced E2 protein on immunoblots. Three epitopes were detected with polyclonal anti-SFV serum at amino acid positions 135 to 141, 177 to 185 and 240 to 246 of the E2 protein. Synthetic peptides containing these epitopes were coupled to a carrier protein and tested as a vaccine. Mice immunized with the peptide containing amino acids 240 to 255 of protein E2 were protected against a challenge with virulent SFV but protection of mice immunized with the peptides containing amino acids 126 to 141 or 178 to 186 was only marginally better than that of controls. The prechallenge sera of most peptide-immunized mice reacted with SFV-infected cells but none of these sera neutralized the virus in vitro. However, protection of mice correlated well with SFV-specific antibody titre, suggesting antibody-mediated protection.

Varying role of alpha/beta interferon in the antiviral efficacy of synthetic immunomodulators against Semliki Forest virus infection.

The question of whether interferon alpha/beta is the common mechanism of antiviral action of synthetic immunomodulators was investigated in B6C3F1 mice infected with Semliki Forest virus. Mice were treated with various concentrations of normal sheep serum or potent anti-alpha/beta interferon antiserum, inoculated with the immunomodulators, and infected 24 hours later with virus. Three patterns emerged. The antiviral action of the pyrimidinone (ABMP) and the oral interferon inducer (CL246,738) appeared to be mediated primarily by interferon alpha/beta; their protective ability was almost completely abrogated by treatment with low levels of anti-alpha/beta interferon antiserum. The antiviral action of two other immunomodulators, a mismatched polyribonucleotide (Ampligen) and a polyanionic copolymer (MVE-2) at least partially involved interferon. Activity of these compounds was reduced, but not consistently eliminated by treatments with high doses of antiserum. The antiviral activity of another polyribonucleotide, polyriboinosinic-cytidylic acid complexed with lysine carboxymethylcellulose (poly ICLC), was not affected by treatment with even the highest amount of antiserum (two injections of 100,000 neutralizing units each). Almost complete protection by poly ICLC was observed despite the fact that this high concentration of antiserum, when given alone, caused a decrease in natural resistance to Semliki Forest virus infection. Taken together, these results indicate that induction of interferon alpha/beta does not appear to be the major common mechanism of antiviral activity among these diverse synthetic immunomodulators.

A protective monoclonal anti-idiotypic vaccine to lethal Semliki Forest virus infection in BALB/c mice.

Two monoclonal anti-idiotypic antibodies (ab2 MAbs), designated 1.13A112 (immunoglobulin G type 2a [IgG2a]) and 1.13A321 (IgG1), were prepared against Semliki Forest virus (SFV)-neutralizing ab1 MAb UM 1.13. They were identified in hybridoma supernatant fluid by their capacity to block UM 1.13-mediated neutralization of SFV. Although the neutralization-blocking capacities of the ab2 MAbs did not differ, only 1.13A321 evoked SFV-neutralizing ab3 antibodies upon intracutaneous and subcutaneous immunization of BALB/c mice with 1.13A321 chemically cross-linked to keyhole limpet hemocyanin and combined with the adjuvant Quil A. SFV-neutralizing ab3 antibodies appeared in serum within 10 days after primary immunization, and neutralizing antibody titers could be as high as 1/1,000 at day 35. All mice who had developed SFV-neutralizing antibodies upon anti-idiotypic immunization survived an otherwise lethal challenge with virulent SFV. However, induction of SFV-neutralizing ab3 antibodies by ab2 MAb 1.13A321 proved to be genetically restricted to BALB/c mice; even haplotype-identical (H-2d) DBA/2 mice did not respond, and consequently those animals died after infection with virulent SFV.

Induced sero-conversion in heifers with a field strain of bovine pestivirus--a comparison of methods and doses.

Losses from pestivirus infection in a closed herd of cattle occurred over several years. In order to prevent further losses, controlled exposure of non-pregnant heifers to pestivirus from viraemic carrier animals was undertaken. Two initial experiments were conducted using either intra-nasal EDTA blood or field contact. Subsequently, other yearling heifers were inoculated with various dilutions of serum using subcutaneous, conjunctival and intra-nasal routes. Effective doses were determined. Neither inoculation nor contact infection produced any clinical illness. The highest dilutions of serum at which sero-conversion occurred were conjunctival, undiluted; intranasal, 10(-1) and subcutaneous 10(-5). With the subcutaneous route all heifers sero-converted at 10(-3). The results for the subcutaneous inoculations showed that the 50% infectious dose for cattle was not distinguishable from that determined in cell culture. Inoculation with a field strain of pestivirus in freeze-thawed serum has effectively and safely induced sero-conversion in heifers. Inoculation of all cattle at risk is considered necessary because no secondary transmission from inoculated heifers was observed.

Guanosine analogues. Synthesis of nucleosides of certain 3-substituted 6-aminopyrazolo[3,4-d]pyrimidin-4(5H)-ones as potential immunotherapeutic agents.

Several guanosine analogues were synthesized in the pyrazolo[3,4-d]pyrimidine ring system with various substituents at the 3-position. The new analogues prepared here include the CH3 (2-amino-3-methyl-1-beta-D-ribofuranosylpyrazolo[3,4-d]pyrimidin-4 (5H)-one, 13a), the phenyl (2-amino-3-phenyl-1-beta-D-ribofuranosylpyrazolo[3,4-d]pyrimidin-4 (5H)-one, 13b), and the NH2 (3,6-diamino-1-beta-D-ribofuranosylpyrazolo[3,4-d]pyrimidin-4(5H)- one, 17) substituted derivatives. These new agents, as well as several other 3-substituted derivatives including H, Br, OCH3, COOH, and oxo, were evaluated for their ability to potentiate certain murine immune functions relative to the known active agent 5-amino-3-beta-D-ribofuranosylthiazolo[4,5-d]pyrimidine-2,7(3H,6H) -dione (4, 7-thia-8-oxoguanosine). The biological evaluation included the (1) ex vivo determination of increased natural killer cell function and (2) in vivo antiviral protection against a lethal challenge of Semliki Forest virus. The 3-unsubstituted (5a) and the 3-bromo (5c) derivatives were found to be the most active immunopotentiators in this series.

Studies on the antiviral activity of inorganic heteropolyanions against Semliki forest virus in vivo and vaccinia virus in vitro.

Seven new inorganic heteropolyanions were tested for their antiviral activity. One of these, sodium 12-tungstoborate, was found to protect mice from Semliki forest virus and mouse embryo fibroblast monolayers from vaccinia virus infections. In mice these heteropolyanions exhibited no synergistic antiviral activity with the interferon inducing mycoviral double-stranded RNA.

Ruminant pestivirus infection in pigs.

Ruminant pestivirus infections of pigs have a worldwide distribution. The prevalence is varied and depends mainly on (i) contact with cattle, (ii) age of pigs and (iii) degree of homology of virus strains used for serology, with field strains of bovine virus diarrhoea virus (BVDV) infecting pigs. Emphasis should be laid on sources of BVDV other than cattle, e.g. contaminated vaccines and fetal calf serum. The need for differentiation of pestiviruses (hog cholera, bovine virus diarrhoea and Border disease viruses) is highlighted by the fact that clinical disease syndromes, e.g. growth retardation and wasting, are reminiscent of hog cholera. Monoclonal antibodies are available which differentiate between hog cholera virus (HCV) and ruminant pestiviruses, presumably BVDV. An up-to-date account of the antigenic relationship between pestiviruses is included in the review. Analysis of the in vitro host range of these viruses is considered to be important and may explain infections of pigs with pestiviruses other than HCV. Recent results have shown the existence of "specialists" amongst BVDV strains for bovine cells, and a few isolates also performed well in cultures of the PK15 cell line. In contrast, multipotent BVDV strains presumably have additional attachment sites for ovine and porcine cells. Identification of receptors on ovine and porcine cells could contribute to a clear distinction between BVDV and HCV infections of pigs. Immediate control measures for BVDV infections of pigs are not required. However, such infections may interfere with serologic surveys and surveillance on a herd basis and, therefore, impair eradication programmes and efforts to maintain the status in countries declared free of hog cholera.

Blocking by anti-idiotypic antibodies of monoclonal antibody-mediated protection against lethal Semliki Forest virus in mice.

Semliki Forest virus-(SEV) neutralizing monoclonal antibodies (MoAbs), produced after fusion of spleen cells from BALB/c mice and myeloma cell line P3-X63-AG8. 653 or SP2/0, were used for anti-idiotypic immunization of female BALB/c mice. Two intracutaneous immunizations (2 x 40 micrograms per animal), 3 weeks apart, with keyhole limpet haemocyanin-conjugated MoAbs mixed with the saponin Quil A were sufficient to induce high levels of anti-idiotypic antibodies in the circulation of these mice with the capacity to block specifically in vitro MoAb-mediated virus neutralization. Anti-idiotypic antibodies against SFV-neutralizing MoAbs, either passively transferred or actively acquired by immunization, are also able to abrogate (specifically) passive immunity, mediated by critical protective doses of MoAb, in mice against infection with a lethal strain of SFV. Furthermore we confirmed by intervention with anti-idiotypic serum in vivo that an SFV-neutralizing MoAb exerts its greatest protective effect during the first 2 days of infection.

Semliki Forest virus E2 envelope epitopes induce a nonneutralizing humoral response which protects mice against lethal challenge.

Along the 422 amino acids of the Semliki Forest virus (SFV) E2 envelope glycoprotein, we identified 13 peptide cassettes (ranging in size from 15 to 25 amino acids and designated A through N) that contain hydrophilic sequences flanked by amino acid sequences conserved in the E2 envelopes of the alphavirus family. Six peptide blocks containing either a single cassette or two to three contiguous cassettes (A, BC, DE, FG, HIK, and LMN) were produced in Escherichia coli as recombinant proteins fused to the N terminus of beta-galactosidase. All of the SFV E2 recombinant polypeptides except A-beta-galactosidase were recognized on Western blots (immunoblots) by anti-SFV polyclonal antisera. In addition, these five recombinant proteins induced in mice antibodies that interacted specifically with SFV E2 protein on Western blots as well as with the intact virions in an enzyme-linked immunosorbent assay. The six hybrid proteins were used to vaccinate mice and were tested for the ability to confer resistance against lethal doses of SFV. Peptides BC and HIK, located at amino acid positions 114 to 149 and 216 to 288, respectively, of E2, protected partially (40 to 60%) against SFV challenge. A third peptide, LMN, located between amino acid positions 289 and 352, rendered mice totally resistant to an SFV challenge of 250 50% lethal doses. The partially protective effects of the BC and HIK cassettes and the high efficacy of the LMN cassette were consistently demonstrated, independent of the adjuvant (complete Freund or alum), immunization protocol, and strain of mice used. None of the antisera raised against any given cassette could neutralize the virus in an in vitro tissue culture assay or in a plaque reduction neutralization test. Nevertheless, passive transfer experiments demonstrated that in the case of LMN, the protective effect was mainly of a humoral nature.