Immunogenicity,Protective Efficacy,and Non-Replicative Status of the HSV-2 Vaccine Candidate HSV529 in Mice and Guinea Pigs

HSV-2 vaccine is needed to prevent genital disease, latent infection, and virus transmission. A replication-deficient mutant virus (dl5-29) has demonstrated promising efficacy in animal models of genital herpes. However, the immunogenicity, protective efficacy, and non-replicative status of the highly purified clinical vaccine candidate (HSV529) derived from dl5-29 have not been evaluated. Humoral and cellular immune responses were measured in mice and guinea pigs immunized with HSV529. Protection against acute and recurrent genital herpes, mortality, latent infection, and viral shedding after vaginal HSV-2 infection was determined in mice or in naïve and HSV-1 seropositive guinea pigs. HSV529 replication and pathogenicity were investigated in three sensitive models of virus replication: severe combined immunodeficient (SCID/Beige) mice inoculated by the intramuscular route, suckling mice inoculated by the intracranial route, and vaginally-inoculated guinea pigs. HSV529 immunization induced HSV-2-neutralizing antibody production in mice and guinea pigs. In mice, it induced production of specific HSV-2 antibodies and splenocytes secreting IFNγ or IL-5. Immunization effectively prevented HSV-2 infection in all three animal models by reducing mortality, acute genital disease severity and frequency, and viral shedding. It also reduced ganglionic viral latency and recurrent disease in naïve and HSV-1 seropositive guinea pigs. HSV529 replication/propagation was not detected in the muscles of SCID/Beige mice, in the brains of suckling mice, or in vaginal secretions of inoculated guinea pigs. These results confirm the non-replicative status, as well as its immunogenicity and efficacy in mice and guinea pigs, including HSV-1 seropositive guinea pigs. In mice, HSV529 produced Th1/Th2 characteristic immune response thought to be necessary for an effective vaccine. These results further support the clinical investigation of HSV529 in human subjects as a prophylactic vaccine.     

Regulation by recombinant interleukin-2 of protective immunity against recurrent herpes simplex virus type 2 genital infection in guinea pigs.

The goal of our study was to determine whether recombinant interleukin-2 (rIL-2) could modify the recurrence pattern of chronic herpes simplex virus type 2 (HSV-2) genital infection in guinea pigs. Animals that developed symptomatic acute HSV-2 infection were distributed at 14 days after viral inoculation into several treatment groups, which were similar with respect to the severity of acute disease. Three rIL-2 dosages administered for 4 weeks in daily subcutaneous injections were tested in this study: 5 X 10(3), 5 X 10(4), and 2.5 X 10(5) U. Daily observations of the animals showed a significant decrease of the incidence of new recurrent lesions with the use of 5 X 10(4) U of rIL-2 (rate of recurrence, 0.08, compared with 0.21 in untreated controls), whereas the other rIL-2 regimens did not affect the overall rate of recurrence. Weekly analysis of recurrences showed that treatment with 5 X 10(4) U of rIL-2 was effective only during the first 3 weeks of use and that 2.5 X 10(5) U of rIL-2 markedly decreased the rate of recurrence in the first week of treatment but not in subsequent weeks. The loss of clinical protection in both groups coincided with the production of neutralizing antibodies to rIL-2. The immune mechanisms possibly involved in the protective effect of rIL-2 in chronic HSV-2 disease were further investigated. Production of gamma interferon correlated well with clinical protection, and circulating levels dropped at the time when neutralizing antibodies to rIL-2 developed. Nonspecific cytotoxicity represented by natural killer cell and lymphokine-activated killer cell activities was also increased in the treated guinea pigs. Antibody titers and lymphocyte proliferation to herpes simplex antigen were similar in rIL-2 and placebo recipients. Finally, we found that the rIL-2-induced immune stimulation was as protective against recurrent HSV-2 disease in guinea pigs as the viral suppression achieved with acyclovir. However, the biological activity of both drugs was not additive when they were coadministered.     

Herpes simplex virus type 2 UL24 gene is a virulence determinant in murine and guinea pig disease models

A herpes simplex virus type 2 (HSV-2) UL24 beta-glucuronidase (UL24-betagluc) insertion mutant was derived from HSV-2 strain 186 via standard marker transfer techniques. Cell monolayers infected with UL24-betagluc yielded cytopathic effect with syncytium formation. UL24-betagluc replicated to wild-type viral titers in three different cell lines. UL24-betagluc was not virulent after intravaginal inoculation of BALB/c mice in that all inoculated animals survived doses up to 400 times the 50% lethal dose (LD50) of the parental virus. Furthermore, few UL24-betagluc-inoculated mice developed any vaginal lesions. Intravaginal inoculation of guinea pigs with UL24-betagluc at a dose equivalent to the LD50 of parental virus (approximately 5 x 10(3) PFU) was not lethal (10/10 animals survived). Although genital lesions developed in some UL24-betagluc-inoculated guinea pigs, both the overall number of lesions and the severity of disease were far less than that observed for animals infected with parental strain 186.     

Adenovirus-mediated RNA interference against foot-and-mouth disease virus infection both in vitro and in vivo

Foot-and-mouth disease virus (FMDV) infection is responsible for the heavy economic losses in stockbreeding each year. Because of the limited effectiveness of existing vaccines and antiviral drugs, the development of new strategies is needed. RNA interference (RNAi) is an effective means of suppressing virus replication in vitro. Here we demonstrate that treatment with recombinant, replication-defective human adenovirus type 5 (Ad5) expressing short-hairpin RNAs (shRNAs) directed against either structural protein 1D (Ad5-NT21) or polymerase 3D (Ad5-POL) of FMDV totally protects swine IBRS-2 cells from homologous FMDV infection, whereas only Ad5-POL inhibits heterologous FMDV replication. Moreover, delivery of these shRNAs significantly reduces the susceptibility of guinea pigs and swine to FMDV infection. Three of five guinea pigs inoculated with 10(6) PFU of Ad5-POL and challenged 24 h later with 50 50% infectious doses (ID50) of homologous virus were protected from the major clinical manifestation of disease: the appearance of vesicles on the feet. Two of three swine inoculated with an Ad5-NT21-Ad5-POL mixture containing 2 x 10(9) PFU each and challenged 24 h later with 100 ID50 of homologous virus were protected from the major clinical disease, but treatment with a higher dose of adenovirus mixture cannot promote protection of animals. The inhibition was rapid and specific because treatment with a control adenovirus construct (Ad5-LacZ) expressing Escherichia coli galactosidase-specific shRNA showed no marked antiviral activity. Our data highlight the in vivo potential of RNAi technology in the case of FMD.     

Use of immunostimulatory sequence-containing oligonucleotides as topical therapy for genital herpes simplex virus type 2 infection

Synthetic oligonucleotides containing CpG motifs in specific sequence contexts have been shown to induce potent immune responses. We have evaluated mucosal administration of two immunostimulatory sequence (ISS)-containing phosphorothioate-stabilized oligonucleotides for antiherpetic efficacy in animal models. The ISS oligonucleotides, suspended in phosphate-buffered saline, were tested in mouse and guinea pig vaginal models of herpes simplex virus type 2 (HSV-2) infection. For comparison, groups of untreated, non-ISS oligonucleotide-treated, and acyclovir-treated animals also were monitored. The results indicated that vaginal epithelial application of ISS (up to 6 h after viral inoculation) with mice lethally challenged with HSV-2 delayed disease onset and reduced the number of animals that developed signs of disease (P = 0.003). ISS application significantly increased survival rates over those of controls (P = 0.0014). The ISS also impacted an established infection in the guinea pig model of HSV-2 disease. A single administration of ISS (21 days after viral inoculation) significantly reduced the frequency and severity of HSV-2 lesions compared to results with non-ISS oligonucleotide-treated and untreated guinea pigs (P < 0.01). HSV-2 is shed from the vaginal cavity of the guinea pig in the absence of lesions, similar to the case with humans. As an additional indication of ISS efficacy, the magnitude of viral shedding also was significantly reduced in ISS-treated animals (P < 0.001). These effects appeared to be immunologically mediated, since ISS had no direct effect on HSV-2 replication in vitro using standard plaque assays. These data suggest that ISS may be useful in the treatment and control of genital herpes in humans.     

Complement-requiring neutralizing antibody in guinea pigs with primary and recurrent genital herpes

Guinea pigs inoculated intravaginally with herpes simplex virus type 2 (HSV-2) strain 1868 produced a serum complement-requiring neutralizing (CRN) antibody during primary acute infection, i.e., 10 days postinoculation. The CRN antibody titers in the guinea pig sera decreased to less than 1:10 after heating at 56 degrees C for 30 min. It was found that 32 units of complement were necessary to obtain a satisfactory HSV-2 neutralizing antibody titer. Nonheated sera significantly reduced virus infectivity titers when mixed with 3.5 log10 PFU of HSV-2 and incubated at 37 degrees C for 20 to 60 min (P less than 0.001), whereas the same sera after heating at 56 degrees C for 30 min showed no inhibitory effect. Only 27.3% of infected guinea pigs had low serum non-CRN antibody titers ranging from 1:20 to 1:40. In addition, no evidence of increase in CRN antibody titers was noted during spontaneous recurrent genital herpes infection.     

Glycosaminoglycans are not indispensable for the anti-herpes simplex virus type 2 activity of lactoferrin

Lactoferrin has been recognized as a potent inhibitor of human herpetic viruses, such as herpes simplex type 1 (HSV-1) and 2 (HSV-2). In particular, bovine lactoferrin (bLf) has been found to prevent viral infection by binding to heparan sulphate (HS) glycosaminoglycans (GAGs) that in turn can act as cell receptors for human herpetic viruses. In this study we further investigate the mechanism of inhibiting activity of both human lactoferrin (hLf) and bLf against HSV-2. The antiviral effect of these proteins towards HSV-2 strain 333 and its glycoprotein C (gC)-truncated derivative HSV-2 gC-neg1 has been tested in monkey kidney cells. Our results indicate that the antiviral activity of bLf does not involve gC-HS interaction as there was no difference in its effectiveness towards wild type and mutant virus. As regards hLf, the mutant virus HSV-2 gC-neg1 was more sensitive compared to the wild type, suggesting that the human protein might interact with some viral structures that in wild-type viruses are masked by gC. When the modulation of HSV-2 infection by bLf and hLf was investigated under different experimental conditions, the bovine protein proved more effective than the human protein. Moreover, we found that, differently from what observed with HSV-1, bLf inhibited HSV-2 plaque-forming activity also in cells devoid of GAG expression. These results suggest that bLf may block a virus receptor of non-GAG nature and add new information on the anti-herpes virus activity of this protein, confirming it as an outstanding candidate for the treatment of herpetic infections.     

Immunization with herpes simplex virus 2 (HSV-2) genes plus inactivated HSV-2 is highly protective against acute and recurrent HSV-2 disease

To date, no vaccine that is safe and effective against herpes simplex virus 2 (HSV-2) disease has been licensed. In this study, we evaluated a DNA prime-formalin-inactivated-HSV-2 (FI-HSV2) boost vaccine approach in the guinea pig model of acute and recurrent HSV-2 genital disease. Five groups of guinea pigs were immunized and intravaginally challenged with HSV-2. Two groups were primed with plasmid DNAs encoding the secreted form of glycoprotein D2 (gD2t) together with two genes required for viral replication, either the helicase (UL5) and DNA polymerase (UL30) genes or the single-stranded DNA binding protein (UL29) and primase (UL52) genes. Both DNA-primed groups were boosted with FI-HSV2 formulated with monophosphoryl lipid A (MPL) and alum adjuvants. Two additional groups were primed with the empty backbone plasmid DNA (pVAX). These two groups were boosted with MPL and alum (MPL-alum) together with either formalin-inactivated mock HSV-2 (FI-Mock) or with FI-HSV2. The final group was immunized with gD2t protein in MPL-alum. After challenge, 0/9 animals in the group primed with UL5, UL30, and gD2t DNAs and all 10 animals in the mock-immunized control group (pVAX-FI-Mock) developed primary lesions. All mock controls developed recurrent lesions through day 100 postchallenge. Only 1 guinea pig in the group primed with pVAX DNA and boosted with FI-HSV2 (pVAX-FI-HSV2 group) and 2 guinea pigs in the group primed with UL5, UL30, and gD2t DNAs and boosted with FI-HSV2 (UL5, UL30, gD2t DNA-FI-HSV2 group) developed recurrent lesions. Strikingly, the UL5, UL30, gD2t DNA-FI-HSV2 group showed a 97% reduction in recurrent lesion days compared with the mock controls, had the highest reduction in days with recurrent disease, and contained the lowest mean HSV-2 DNA load in the dorsal root ganglia.     

The cotton rat (Sigmodon hispidus) is a permissive small animal model of human metapneumovirus infection, pathogenesis, and protective immunity

Human metapneumovirus (hMPV) is a newly described paramyxovirus that is an important cause of acute respiratory tract disease. We undertook to develop a small animal model of hMPV infection, pathogenesis, and protection. Hamsters, guinea pigs, cotton rats, and nine inbred strains of mice were inoculated intranasally with hMPV. The animals were sacrificed, and nasal and lung tissue virus yields were determined by plaque titration. None of the animals exhibited respiratory symptoms. The quantity of virus present in the nasal tissue ranged from 4.6 x 10(2) PFU/gram tissue (C3H mice) to greater than 10(5) PFU/gram (hamster). The amount of virus in the lungs was considerably less than in nasal tissue in each species tested, ranging from undetectable (<5 PFU/g; guinea pigs) to 1.8 x 10(5) PFU/gram (cotton rat). The peak virus titer in cotton rat lungs occurred on day 4 postinfection. hMPV-infected cotton rat lungs examined on day 4 postinfection exhibited histopathological changes consisting of peribronchial inflammatory infiltrates. Immunohistochemical staining detected virus only at the luminal surfaces of respiratory epithelial cells throughout the respiratory tract. hMPV-infected cotton rats mounted virus-neutralizing antibody responses and were partially protected against virus shedding and lung pathology on subsequent rechallenge with hMPV. Viral antigen was undetectable in the lungs on challenge of previously infected animals. This study demonstrates that the cotton rat is a permissive small animal model of hMPV infection that exhibits lung histopathology associated with infection and that primary infection protected animals against subsequent infection. This model will allow further in vivo studies of hMPV pathogenesis and evaluation of vaccine candidates.     

Recombinant vesicular stomatitis virus vectors expressing herpes simplex virus type 2 gD elicit robust CD4+ Th1 immune responses and are protective in mouse and guinea pig models of vaginal challenge

Recombinant vesicular stomatitis virus (rVSV) vectors offer an attractive approach for the induction of robust cellular and humoral immune responses directed against human pathogen target antigens. We evaluated rVSV vectors expressing full-length glycoprotein D (gD) from herpes simplex virus type 2 (HSV-2) in mice and guinea pigs for immunogenicity and protective efficacy against genital challenge with wild-type HSV-2. Robust Th1-polarized anti-gD immune responses were demonstrated in the murine model as measured by induction of gD-specific cytotoxic T lymphocytes and increased gamma interferon expression. The isotype makeup of the serum anti-gD immunoglobulin G (IgG) response was consistent with the presence of a Th1-CD4+ anti-gD response, characterized by a high IgG2a/IgG1 IgG subclass ratio. Functional anti-HSV-2 neutralizing serum antibody responses were readily demonstrated in both guinea pigs and mice that had been immunized with rVSV-gD vaccines. Furthermore, guinea pigs and mice were prophylactically protected from genital challenge with high doses of wild-type HSV-2. In addition, guinea pigs were highly protected against the establishment of latent infection as evidenced by low or absent HSV-2 genome copies in dorsal root ganglia after virus challenge. In summary, rVSV-gD vectors were successfully used to elicit potent anti-gD Th1-like cellular and humoral immune responses that were protective against HSV-2 disease in guinea pigs and mice.     

Immunization with recombinant varicella-zoster virus expressing herpes simplex virus type 2 glycoprotein D reduces the severity of genital herpes in guinea pigs.

Varicella-zoster virus (VZV) is an attractive candidate for a live-virus vector for the delivery of foreign antigens. The Oka vaccine strain of VZV is safe and effective in humans, and recombinant Oka VZV (ROka) can be generated by transfecting cells with a set of overlapping cosmid DNAs. By this method, the herpes simplex virus type 2 (HSV-2) glycoprotein D (gD2) gene was inserted into an intergenic site in the unique short region of the Oka VZV genome. Expression of gD2 in cells infected with the recombinant Oka strain VZV (ROka-gD2) was confirmed by antibody staining of fixed cells and by immunoblot analysis. Immune electron microscopy demonstrated the presence of gD2 in the envelope of ROka-gD2 virions. The ability of ROka-gD2 to protect guinea pigs against HSV-2 challenge was assessed by inoculating animals with three doses of uninfected human fibroblasts, fibroblasts infected with ROka VZV, or fibroblasts infected with ROka-gD2. Neutralizing antibodies specific for HSV-2 developed in animals immunized with ROka-gD2. Forty days after the third inoculation, animals were challenged intravaginally with HSV-2. Inoculation of guinea pigs with ROka-gD2 significantly reduced the severity of primary HSV-2 infection (P < 0.001). These experiments demonstrate that the Oka strain of VZV can be used as a live virus vector to protect animals from disease with a heterologous virus.     

Regulation of guinea-pig immune functions by interleukin 2: critical role of natural killer activity in acute HSV-2 genital infection

We have previously demonstrated that recombinant interleukin 2 (rIL 2) has a protective effect against acute HSV-2 infection in guinea pigs with a biphasic dose response which peaked between 4 and 20 X 10(4) U/kg, whereas 8 X 10(5) U/kg showed no effect on disease. Animals that escaped infection appeared lack immunologic memory to HSV-2, suggesting a nonspecific immune mechanism. In this study we have found that NK activity of fresh splenocytes measured against HSV-2 infected human foreskin fibroblast (HFF) is stimulated in vitro and in vivo by rIL 2 in a biphasic dose range similar to that determined for protection against disease. In contrast, lymphokine-activated killer (LAK)-mediated lysis of P815 showed a linear response to increasing concentrations of rIL 2 both in vitro and in vivo. Transfer of LAK cells did not alter the rate of infection after HSV-2 challenge. Anti-asialo GM-1 eliminated rIL 2 protection against HSV-2 infection. It also blocked HSV-2/HFF lysis and partially decreased P815 lysis in vitro; however, in vivo it inhibited both natural killer (NK) activity and LAK generation, failing to distinguish which of the lytic cells was responsible for the effect against infection. Early IgG production (7 days post-infection) was enhanced by rIL 2 administration before viral inoculation, but it did not influence the rate of infection as compared with controls. Polyclonal IgM secretion was not found to play a role in acute protection. Circulating serum interferon levels were enhanced with increasing concentrations of rIL 2 but did not correlate with the biphasic dose curve for protection. Therefore of these mechanisms the one that is most closely related to the protective effect of rIL 2 against primary HSV-2 infection appears to be NK-mediated lysis, although the other mechanisms may add to this effect.     

Early adrenal infection by herpes simplex virus type-1 (Miyama + GC strain): special reference to inoculation dose and spread from the adrenal to the central nervous system

Male C3H/HeN mice, aged 5 weeks, were inoculated intraperitoneally (i.p.) with different doses (1 x 10(3), 1 x 10(5), 5 x 10(5), 1 x 10(6) pfu) of the herpes simplex virus type-1 (HSV-1) (Miyama + GC strain). The LD50 of this virus was 10(2) pfu (i.p.) per mouse. All the mice in each group died 12 days after inoculation. Adrenal necrosis was found to be dose-dependent, the threshold dose being 5 x 10(5) pfu. In addition, encephalitis and inflammatory cell infiltration in abdominal ganglia appeared in 3-4 days after inoculation. By the plaque method, HSV-1 was detected first in the adrenal glands, then in neurons in the spinal cord and the brain. These findings suggest that in mice inoculated with doses of virus sufficient to infect the adrenal gland, HSV-1 spreads to the central nervous system through peripheral nerves after replication in the adrenal.     

Characterization of the oncornavirus particles in the plasma of guinea pigs with L2C leukemia.

The inoculation of L2C guinea pig leukemia cells into strain 2 guinea pigs results in the death of the animals within 12 to 15 days. Death is preceded by the simultaneous appearance in the plasma of (i) elevated leukocyte levels, (ii) extracellular virus particles, and (iii) a particle-associated RNA-directed DNA polymerase. This enzyme activity has a cation preference identical to that of the type B bromodeoxyuridine-induced guinea pig virus, i.e., an Mg2+ optimum at 20 mM and no activity using Mn2+. Competitive molecular hybridization studies also revealed that the plasma of leukemic guinea pigs contained approximately 2 X 10(9) genome equivalents per ml of an RNA that is homologous to the RNA of the bromodeoxyuridine-induced guinea pig virus. Morphological observations indicate that most, but not all, of the extracellular particles observed in leukemia plasma are derived from the intracisternal particles seen in the L2C tumor cells. The possibilities that either two viral populations are present or that the in vivo morphogenesis of the type B bromodexoyuridine-inducible guinea pig virus is markedly different from its in vitro morphogenesis are discussed.     

Evaluation of Antiherpetic Compounds Using a Gastric Cancer Cell Line: Pronounced Activity of BVDU against Herpes Simplex Virus Replication

We developed a rapid and simple method for the screening of antiviral agents against herpes simplex virus (HSV) in a model of gastrointestinal herpetic infection in vitro. This method was based on inhibition of HSV-induced cytopathogenicity in gastric adenocarcinoma MKN-28 cells, as monitored by an MTT colorimetric assay. From the various compounds that were evaluated for their activity against HSV-1 and HSV-2, brivudine (BVDU) emerged as the most effective. When the 50% effective concentration (EC₅₀) values of the antiherpes agents in MKN-28 cells were compared with those in human embryo lung MRC-5 cells, all compounds, except for BVDU, showed higher EC₅₀ values in MKN-28 cells. For BVDU the EC₅₀ values in MKN-28 cells were 0.8 (HSV-1) and 0.036 (HSV-2) times the EC₅₀ values in MRC-5 cells. Thus BVDU was 27.5 times more active against HSV-2 in MKN-28 cells than in MRC-5 cells. The MKN-28 gastric cancer cells may be useful for the rapid screening of anti-HSV agents and, in particular, those that may be useful in therapy of gastrointestinal HSV infections in gastrointestinal herpetic infection.     

Porcine conceptus proteins: antiviral activity and effect on 2',5'-oligoadenylate synthetase.

Interferon-like proteins synthesized by conceptuses of domestic ruminants inhibit luteolysis during early pregnancy. Although pig conceptuses secrete trophoblast interferons during the period of CL maintenance, estrogen is involved with maintenance of the CL. The principal purposes of this work were to confirm production of trophoblast interferons by porcine conceptuses and to compare the effect of trophoblast interferons on endometrium of pigs and cattle. When measured using Madin-Darby bovine kidney (MDBK) cells challenged with vesicular stomatitis virus, antiviral activity in uterine flushings from cyclic gilts was not detectable throughout the estrous cycle; however, in pregnant gilts, antiviral activity increased from undetectable amounts to 4-11 x 10(3) U on Days 14, 16, and 18. Porcine embryos in culture produced 1,100 U/embryo/ml/24 h. Porcine conceptus secretory proteins induced 2',5'-oligo(A) synthetase in MDBK cells and in endometrial explants of cows but had no measurable effect on 2',5'-oligo(A) synthetase activity of endometrial explants of pigs. Similarly, endometrial 2',5'-oligo(A) synthetase of pregnant pigs was unaffected in vivo during the period of maximal synthesis of conceptus secretory proteins. Porcine conceptus secretory proteins produced no detectable increase in serum antiviral activity or 2',5'-oligo(A) synthetase activity of blood mononuclear leukocytes in utero-ovarian venous blood. These results suggest that conceptus interferons of pigs play different roles in the establishment of pregnancy compared to their roles in ruminants.     

Genetic mapping of lymphocytic choriomeningitis virus pathogenicity: virulence in guinea pigs is associated with the L RNA segment.

The Armstrong CA 1371 (ARM) and WE strains of lymphocytic choriomeningitis virus (LCMV) differ in the ability to produce disease in adult guinea pigs. Infection with the ARM strain is not lethal, even at high virus doses (greater than 10,000 PFU), whereas the WE strain causes 100% mortality even at low doses (less than 10 PFU). To determine the genetic basis of this virulence, intertypic reassortants were made between the ARM and WE strains of LCMV. The two reassortants with the genotypes WE/ARM (L segment of WE and S segment of ARM) and ARM/WE (L segment of ARM and S segment of WE) were tested for their pathogenicity in guinea pigs. The ARM/WE reassortant was avirulent like the ARM/ARM parental strain. Minimal viral replication was observed in organs of guinea pigs inoculated with 10(2) or 10(5) PFU of ARM/ARM or ARM/WE, and all animals survived. In contrast, the WE/ARM reassortant was highly virulent like the WE/WE parental strain and killed all of the infected animals. High levels of viral replication were observed in guinea pigs infected with the latter two strains. In contrast to these in vivo observations, both the parental strains and the ARM/WE or WE/ARM reassortants had similar growth potential in cultured guinea pig fibroblasts. Thus, the L RNA segment of LCMV WE is important for viral replication in vivo and is associated with fatal acute disease after infection of adult guinea pigs.     

Prevention of lethal respiratory vaccinia infections in mice with interferon-alpha and interferon-gamma

The antiviral efficacy of interferons (IFNs) was evaluated using a vaccinia intranasal infection model in mice in this study. We provide evidence that intranasal administration of IFN-alpha and IFN-gamma (days -1 to +3) resulted in 100 and 90% survival against a lethal respiratory vaccinia infection (8 LD50) in mice, respectively; whereas no animals in the placebo group survived through the study period (21 days). The IFN treatment consisted of a single daily dose of 5x10(3) U per mouse for 5 consecutive days. The efficacy of IFN-gamma was evident even when the IFN-gamma treatments started 1-2 days after infection and when a lower dose (2x10(3) U per mouse) was used. The treatment of IFN-alpha and IFN-gamma reduced the virus titers in the lungs of infected mice by 1000-10,000-fold, when the administration started 1 day after infection. Our data suggest that IFN-alpha and IFN-gamma are effective in protecting vaccinia-infected mice from viral replication in lungs and mortality, and may be beneficial in other human orthopoxvirus infections.     

Live attenuated herpes simplex virus 2 glycoprotein E deletion mutant as a vaccine candidate defective in neuronal spread

A herpes simplex virus 2 (HSV-2) glycoprotein E deletion mutant (gE2-del virus) was evaluated as a replication-competent, attenuated live virus vaccine candidate. The gE2-del virus is defective in epithelial cell-to-axon spread and in anterograde transport from the neuron cell body to the axon terminus. In BALB/c and SCID mice, the gE2-del virus caused no death or disease after vaginal, intravascular, or intramuscular inoculation and was 5 orders of magnitude less virulent than wild-type virus when inoculated directly into the brain. No infectious gE2-del virus was recovered from dorsal root ganglia (DRG) after multiple routes of inoculation; however, gE2-del DNA was detected by PCR in lumbosacral DRG at a low copy number in some mice. Importantly, no recurrent vaginal shedding of gE2-del DNA was detected in immunized guinea pigs. Intramuscular immunization outperformed subcutaneous immunization in all parameters evaluated, although individual differences were not significant, and two intramuscular immunizations were more protective than one. Immunized animals had reduced vaginal disease, vaginal titers, DRG infection, recurrent genital lesions, and recurrent vaginal shedding of HSV-2 DNA; however, protection was incomplete. A combined modality immunization using live virus and HSV-2 glycoprotein C and D subunit antigens in guinea pigs did not totally eliminate recurrent lesions or recurrent vaginal shedding of HSV-2 DNA. The gE2-del virus used as an immunotherapeutic vaccine in previously HSV-2-infected guinea pigs greatly reduced the frequency of recurrent genital lesions. Therefore, the gE2-del virus is safe, other than when injected at high titer into the brain, and is efficacious as a prophylactic and immunotherapeutic vaccine.     

Effects of Oxyamylose and Polyacrylic Acid on Foot-and-Mouth Disease and Hog Cholera Virus Infections

Two interferon-inducing polycarboxylates were tested for antiviral activity on foot-and-mouth disease (FMD) virus infections in mice, guinea pigs, and swine. Polyacrylic acid, given intraperitoneally, had a protective effect on infection by FMD virus administered in the peritoneal cavity of mice and in the foot pad of guinea pigs. Chlorite-oxidized oxyamylose (COAM) was effective in mice at a dosage of 2 mg/kg. Swine were not protected against naturally transmitted FMD by 120 mg/kg of COAM nor by polyacrylic acid. Swine were not totally unresponsive to COAM since it delayed symptoms of hog cholera. Interferon was not detected in the serum of COAM-treated swine. With FMD virus, an example was found of activity of interferon inducers in experimental hosts and lack of activity in a natural host.