Immunization with baculovirus-expressed recombinant rotavirus proteins VP1, VP4, VP6, and VP7 induces CD8+ T lymphocytes that mediate clearance of chronic rotavirus infection in SCID mice.

Clearance of chronic murine rotavirus infection in SCID mice can be demonstrated by adoptive transfer of immune CD8+ T lymphocytes from histocompatible donor mice immunized with a murine homotypic rotavirus (T. Dharakul, L. Rott, and H.B. Greenberg, J. Virol 64:4375-4382, 1990). The present study focuses on the protein specificity and heterotypic nature of cell-mediated clearance of chronic murine rotavirus infection in SCID mice. Heterotypic cell-mediated clearance was demonstrated in SCID mice infected with EDIM (murine) rotavirus after adoptive transfer of CD8+ T lymphocytes from BALB/c mice that were immunized with a variety of heterologous (nonmurine) rotaviruses including Wa (human, serotype 1), SA11 and RRV (simian, serotype 3), and NCDV and RF (bovine, serotype 6). This finding indicates the serotypic independence of T-cell-mediated rotavirus clearance. To further identify the rotavirus proteins that are capable of generating CD8+ T cells that mediate virus clearance, donor mice were immunized with SF-9 cells infected with a baculovirus recombinant expressing one of the following rotavirus proteins: VP1, VP2, NS53 (from RF), VP4, VP7, NS35 (from RRV), VP6, and NS28 (from SA11). SCID mice stopped shedding rotavirus after receiving CD8+ T cells from mice immunized with VP1, VP4, VP6, and VP7 but not with VP2, NS53, NS35, NS28, or wild-type baculovirus. These results suggest that heterotypic cell-mediated clearance of rotavirus in SCID mice is mediated by three of the major rotavirus structural proteins and by a putative polymerase protein.     

Recovery from chronic rotavirus infection in mice with severe combined immunodeficiency: virus clearance mediated by adoptive transfer of immune CD8+ T lymphocytes.

Severe combined immunodeficient (SCID) mice lack both functional T and B cells. These mice develop chronic rotavirus infection following an oral inoculation with the epizootic diarrhea of infant mice (EDIM) rotavirus. Reconstitution of rotavirus-infected SCID mice with T lymphocytes from immunocompetent mice allows an evaluation of a role of T-cell-mediated immunity in clearing chronic rotavirus infection. Complete rotavirus clearance was demonstrated in C.B-17/scid mice 7 to 9 days after the transfer of immune CD8+ splenic T lymphocytes from histocompatible BALB/c mice previously immunized intraperitoneally with the EDIM-w strain of murine rotavirus. The virus clearance mediated by T-cell transfer was restricted to H-2d-bearing T cells and occurred in the absence of rotavirus-specific antibody as determined by enzyme-linked immunosorbent assay, neutralization, immunohistochemistry, and radioimmunoprecipitation. Temporary clearance of rotavirus was observed after the transfer of immune CD8+ T cells isolated from the intestinal mucosa (intraepithelial lymphocytes [IELs]) or the spleens of BALB/c mice previously infected with EDIM by the oral route. Chronic virus shedding was transiently eliminated 7 to 11 days after spleen cell transfer and 11 to 12 days after IEL transfer. However, recurrence of rotavirus infection was detected 1 to 8 days later in all but one SCID recipient receiving cells from orally immunized donors. The viral clearance was mediated by IELs that were both Thy1+ and CD8+. These data demonstrated that the clearance of chronic rotavirus infection in SCID mice can be mediated by immune CD8+ T lymphocytes and that this clearance can occur in the absence of virus-specific antibodies.     

Extramucosal spread and development of hepatitis in immunodeficient and normal mice infected with rhesus rotavirus.

The pathogenic profiles of two heterologous animal rotaviruses, rhesus rotavirus strain MMU 18006 and bovine rotavirus strain WC3, were evaluated in mice with severe combined immunodeficiency (SCID mice) and normal BALB/c mice. Control animals were inoculated with homologous murine strain EDIM 5099 or a tissue culture-adapted murine rotavirus. Heterologous infection with rhesus rotavirus resulted in hepatitis in 84% of SCID and 21% of BALB/c mice, with mortality rates of 27 and 0%, respectively. Surviving SCID animals developed chronic liver disease, while symptoms in BALB/c mice resolved in 2 to 4 weeks after onset. Histopathologic examination revealed a diffuse hepatitis with focal areas of parenchymal necrosis. Rotavirus was detected in liver tissue from 100% of 29 SCID and 85% (11 of 13) BALB/c animals tested by cell culture infectivity, immunofluorescence, or electron microscopy. No extramucosal spread of virus or hepatitis was observed after infection with heterologous bovine strain WC3 or homologous murine rotaviruses. This finding of a novel rotavirus-induced disease manifestation suggests altered tissue tropism in a heterologous host for a group of viruses previously shown to replicate exclusively in the gut mucosa. The implications of our observations suggest that in human vaccine trials utilizing heterologous rotavirus strains, special attention should be paid to children with immunodeficiency disorders, and screening for hepatic function should be included in vaccine protocols.     

Evidence that resolution of rotavirus infection in mice is due to both CD4 and CD8 cell-dependent activities.

The effector functions responsible for resolution of shedding in mice orally inoculated with the murine rotavirus strain EDIM were identified in B-cell-deficient and normal BALB/c mice after monoclonal antibody (MAb) depletion of CD4 and CD8 cells. When depleted of CD8 cells, B-cell-deficient muMt mice resolved their infections more slowly than nondepleted animals, but CD4 cell depletion caused chronic, high-level shedding. This finding indicated that CD4 cell-dependent immunological effectors other than, or in addition to, CD8 cells played roles in rotavirus resolution in muMt mice in the absence of antibody. The roles of CD4 and CD8 cells in resolution of rotavirus shedding were further characterized in immunologically normal BALB/c mice. Depletion of CD4 cells before EDIM inoculation resulted in rapid resolution of most shedding, but chronic, low-level shedding continued for weeks. When the CD4 cell-depleted BALB/c mice were subsequently depleted of CD8 cells, shedding levels increased significantly (P < 0.001), indicating that CD8 cells were responsible for the rapid but incomplete suppression of rotavirus shedding. Further experimentation revealed that little rotavirus antibody was made in CD4 cell-depleted BALB/c mice, and only after CD4 cells were repopulated did antibody production increase and virus shedding fully resolve. Thus, resolution of rotavirus shedding in both muMt and BALB/c mice was associated with CD4 and CD8 cell effector activities.     

Inhibitory effects of bile acids and synthetic farnesoid X receptor agonists on rotavirus replication

Rotaviruses (group A rotaviruses) are the most important cause of severe gastroenteritis in infants and children worldwide. Currently, an antiviral drug is not available and information on therapeutic targets for antiviral development is limited for rotavirus infection. Previously, it was shown that lipid homeostasis is important in rotavirus replication. Since farnesoid X receptor (FXR) and its natural ligands bile acids (such as chenodeoxycholic acid [CDCA]) play major roles in cholesterol and lipid homeostasis, we examined the effects of bile acids and synthetic FXR agonists on rotavirus replication in association with cellular lipid levels. In a mouse model of rotavirus infection, effects of oral administration of CDCA on fecal rotavirus shedding were investigated. The results demonstrate the following. First, the intracellular contents of triglycerides were significantly increased by rotavirus infection. Second, CDCA, deoxycholic acid (DCA), and other synthetic FXR agonists, such as GW4064, significantly reduced rotavirus replication in cell culture in a dose-dependent manner. The reduction of virus replication correlated positively with activation of the FXR pathway and reduction of cellular triglyceride contents (r(2) = 0.95). Third, oral administration of CDCA significantly reduced fecal virus shedding in mice (P < 0.05). We conclude that bile acids and FXR agonists play important roles in the suppression of rotavirus replication. The inhibition mechanism is proposed to be the downregulation of lipid synthesis induced by rotavirus infection.     

Immunotherapy of cryptosporidiosis in immunodeficient animal models.

Immunotherapy for persistent infection caused by Cryptosporidium parvum was attempted in two immunodeficient animal models. BALB/c Athymic (nude) mice were infected with two oral doses of 2 x 10(7) C. parvum oocysts, and subsequently treated with monoclonal antibody (MAb) 17.41 that neutralizes sporozoites and merozoites. Persistent infection was established in all exposed mice. Daily oral treatment with MAb 17.41 for 10 days significantly reduced (p less than 0.005) the number of C. parvum organisms observed by microscopic study of intestinal tracts of infected mice. Young horses with severe combined immunodeficiency (SCID) also developed persistent infection following oral exposure with 10(8) C. parvum oocysts. In contrast to nude mice, SCID foals exhibited diarrhea associated with oocyst shedding. Two foals were treated orally with MAb 18.44 and immune serum, both of which neutralized C. parvum sporozoites and merozoites. Oocyst shedding patterns did not significantly differ from those in five SCID foals treated with nonimmune reagents. The results obtained indicate that SCID foals are a useful large animal model of clinical disease associated with persistent C. parvum infection, and that nude mice are a convenient animal model for testing therapeutic potential of antibodies in persistent cryptosporidial infection.     

Development of an adult mouse model for studies on protection against rotavirus.

Although mice have been used as an animal model for studies on rotavirus disease, these studies have been limited by the short time period after birth during which mice are susceptible to rotavirus illness (i.e., approximately 15 days). To overcome this limitation, an adult mouse model was developed in which the endpoint was infection rather than illness. The model developed utilized a strain of mouse rotavirus (EDIM) adapted to grow in culture by multiple passages in MA104 cells. The second cell culture passage of EDIM caused severe diarrhea in neonatal BALB/c mice, and little or no amelioration of disease was observed after nine cell culture passages, even when this preparation was plaque purified. Oral administration of 2 x 10(3) PFU of passage 9 also consistently caused infection of mice 4, 10, 15, 30, 60, 120, and 180 days of age as determined by viral shedding and seroconversion. Reinoculation of these mice with the same virus preparation at 2, 3, or 4 months after the first inoculation produced no evidence of reinfection. In contrast, infection of neonatal mice with the heterotypic WC3 bovine rotavirus did not prevent reinfection with culture-adapted EDIM. Thus, this strain of EDIM caused consistent infection of previously uninoculated neonatal and adult BALB/c mice and produced homotypic but not heterotypic protection against reinfection.     

CD8+ T cells can mediate almost complete short-term and partial long-term immunity to rotavirus in mice.

We have recently shown that CD8+ T cells mediate clearance of rotavirus infection in mice. B-cell-deficient J(H)D knockout (-/-) mice depleted of CD8+ T cells become chronically infected with murine rotavirus, and beta2 microglobulin -/- and other mice depleted of CD8+ T cells have a 1- to 4-day delay in clearance of primary rotavirus infection. A role for CD8+ T cells in protection from reinfection with rotavirus was suggested by these studies, because J(H)D -/- mice rechallenged 6 to 8 weeks after primary infection shed smaller quantities of viral antigen and for fewer days than naive mice. Here we show that 8, 11, 13, and 18 days after primary infection the J(H)D -/- mice are almost completely resistant to reinfection and that they are still partially protected from reinfection 6 weeks, 5 months, and 8 months after primary infection. Protection against reinfection was dependent on CD8+ T cells, since J(H)D -/- mice depleted of CD8+ T cells by administration of an anti-CD8 monoclonal antibody became chronically infected with rotavirus upon rechallenge 13 days, 18 days, 6 weeks, and 5 months after primary infection. Thus, CD8+ T cells can actively mediate almost complete short-term and partial long-term protection from reinfection.     

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.     

Restricted virus replication in the spinal cords of nude mice infected with a Theiler''s virus variant.

The Daniels strain of Theiler's murine encephalomyelitis produces a chronic disease which is an animal model for human demyelinating disorders. Previously, we selected a neutralization-resistant virus variant producing an altered and diminished central nervous system disease in immunocompetent mice which was evident during the later stage of infection (after 4 weeks) (A. Zurbriggen and R. S. Fujinami, J. Virol. 63:1505-1513, 1989). The exact epitope determining neurovirulence was precisely mapped to a capsid protein, VP-1, and represents a neutralizing region (A. Zurbriggen, J. M. Hogle, and R. S. Fujinami, J. Exp. Med. 170:2037-2049, 1989). Here, we present experiments with immunoincompetent animals to determine viral replication, spread, and targeting to the central nervous system in the absence of detectable antibodies or functional T cells. Nude mice were infected orally, and the virus was monitored by plaque assay, immunohistochemistry, and in situ hybridization. Early during the infection (1 week), the variant virus induced an acute disease comparable to that induced by the wild-type virus in these nude mice. Alterations in tropism in the central nervous system were not apparent when wild-type parental Daniels strain virus was compared with the variant virus. Moreover, variant virus replicated in tissue culture (BHK-21 cells) to similarly high titers in a time course identical to that of the wild-type virus (A. Zurbriggen and R. S. Fujinami, J. Virol. 63:1505-1513, 1989). However, replication of the variant virus versus the wild-type virus within the spinal cord of athymic nude mice infected per os was substantially restricted by 6 weeks postinfection. Therefore, the reduced neurovirulence in the later stage (6 weeks) of the disease is most likely due to a diminished growth rate or spread of the variant virus in the central nervous system rather than to marked differences in viral tropism.     

Immunomodulatory function of lactic acid bacteria

Using mice, we found that oral administration of Bifidobacterium breve YIT4064 (B. breve) activated the humoral immune system, augmented anti-rotavirus IgA production or anti-influenza virus (IFV) IgG production and protected against rotavirus infection or influenza infection, respectively. Furthermore, when the B. breve was given to infants from an infant home, there was a significant reduction of the frequency of rotavirus shedding in stool samples during the administration of the bacteria. It was also found, again using mice, that oral administration of Lactobacillus casei strain Shirota (LcS) stimulated type 1 helper T (Th1) cells, activated the cellular immune system and inhibited incidence of tumors and IgE production. These results demonstrated that these two strains of lactic acid bacteria modulated the different immune systems each in its own way and prevented against various diseases.     

Group A Rotavirus Infection and Age-Dependent Diarrheal Disease in Rats: a New Animal Model To Study the Pathophysiology of Rotavirus Infection

Group A rotaviruses are major pathogens causing acute gastroenteritis in children and animals. To determine if group A rotavirus replicates and induces disease in rats, antibody-negative Lewis neonatal or adult rats were inoculated orally with tissue culture-adapted human (Wa, WI61, and HAL1166), simian (rhesus rotavirus [RRV] and SA11), bovine (WC3), lapine (ALA), or porcine (OSU) rotavirus strains, wild-type murine (EC(wt)) rotavirus strain, or phosphate-buffered saline (PBS). Rotavirus infection in rats was evaluated by (i) clinical findings, (ii) virus antigen shedding or infectious virus titers in the feces or intestinal contents measured by enzyme-linked immunosorbent assay or fluorescent-focus assay, (iii) histopathological changes in the small intestine, (iv) distribution of rotavirus antigen in small-intestine sections by immunofluorescence, and (v) growth rate. Rotavirus infection of 5-day-old but not > or =21-day-old rats resulted in diarrhea that lasted from 1 to 10 days postinoculation. The severity of disease and spread of infection to naIve littermates differed depending on the virus strain used for inoculation. The duration of virus antigen shedding following infection was considerably prolonged (up to 10 days) in neonatal rats compared to that in 21-day-old rats (1 or 2 days). Based on lack of virus antigen shedding and disease induction, the murine EC(wt) rotavirus was the only strain tested that did not infect rats. Histopathological changes in the small-intestine mucosa of 5-day-old RRV-inoculated rats but not of PBS-inoculated rats was limited to extensive enterocyte vacuolation in the ileum. In RRV-inoculated neonatal rats, rotavirus antigen was detected in the epithelial cells on the upper half of the intestinal villi of the jejunum and ileum. In addition, infection of neonatal rats with RRV but not with PBS resulted in reduced weight gain. Rats infected with group A rotaviruses provide a new animal model with unique features amenable to investigate rotavirus pathogenesis and the molecular mechanisms of intestinal development, including physiological factors that may regulate age-dependent rotavirus-induced diarrhea.     

Successful sanitation of an EDIM-infected mouse colony by breeding cessation

Despite decreasing prevalence, rotavirus infections still rank among the most important viral infections in colonies of laboratory mice. Although the disease is characterized by low mortality and a relatively short and mild clinical period, the infection has the potential to alter the outcome of experiments substantially. For animal facilities, it is therefore essential to eradicate the virus. Here we report a successful sanitation of a rotavirus-infected mouse colony in an animal facility. Despite a high ratio of transgenic and partially immunodeficient strains, a permanent eradication of the virus was achieved by euthanasia of highly susceptible mice, a prolonged breeding cessation in areas containing immunocompromised mice and a strict hygienic management. The management of a rotavirus infection reported here is a feasible and inexpensive opportunity for sanitation that benefits from maintaining most of the animal population, even in today's mouse colonies comprising mainly transgenic mice with unknown or compromised immune status.     

Comparison of mucosal and systemic humoral immune responses and subsequent protection in mice orally inoculated with a homologous or a heterologous rotavirus.

Rotaviruses are the single most important cause of severe diarrhea in young children worldwide, and vaccination is probably the most effective way to control the disease. Most current live virus vaccine candidates are based on the host range-restricted attenuation of heterologous animal rotaviruses in humans. The protective efficacy of these vaccine candidates has been variable. To better understand the nature of the heterologous rotavirus-induced active immune response, we compared the differences in the mucosal and systemic immune responses generated by heterologous (nonmurine) and homologous (murine) rotaviruses as well as the ability of these infections to produce subsequent protective immunity in a mouse model. Sucking mice were orally inoculated with a heterologous simian or bovine rotavirus (strain RRV or NCDV) or a homologous murine rotavirus (wild-type or tissue culture-adapted) strain EHP at various doses. Six weeks later, mice were challenged with a virulent murine rotavirus (wild-type strain ECW) and the shedding of viral antigen in feces was quantitated. Levels of rotavirus-specific serum immunoglobulin G (IgG) and fecal IgA prior to challenge were measured and correlated with subsequent viral shedding or protection. Heterologous rotavirus-induced active protection was highly dependent on the strain and dose of the virus tested. Mice inoculated with a high dose (10(7) PFU per mouse) of RRV were completely protected, while the protection was diminished in animals inoculated with NCDV or lower doses of RRV. The ability of a heterologous rotavirus to stimulate a detectable intestinal IgA response correlated with the ability of the virus to generate protective immunity. Serum IgG titer did not correlate with protection. Homologous rotavirus infection, on the other hand, was much more efficient at inducing both mucosal and systemic immune responses as well as protection regardless of the virulence of the virus strain or the size of the immunizing dose.     

Passive immunity modulates genetic reassortment between rotaviruses in mixedly infected mice.

Genetic reassortment between simian rotavirus SA11 and rhesus rotavirus (RRV) occurs with high frequency following mixed infection of nonimmune suckling mice (J. L. Gombold and R. F. Ramig, J. Virol. 57:110-116, 1986). We examined the effects of passively acquired homotypic or heterotypic immunity on reassortment in vivo. Passively immune suckling mice obtained from dams immune to either serotype 3 simian rotavirus (SA11) or serotype 6 bovine rotavirus (NCDV) were infected orally with either SA11 or RRV or a mixture of SA11 and RRV (both serotype 3 viruses). At various times postinfection, signs of disease were noted and the intestines of individual mice were removed and homogenized for titration of infectious virus and isolation of progeny plaques. Electrophoresis of genomic RNA was used to identify reassortants among the viral progeny isolated from infected animals. No reassortants (less than 0.45%) were detected among 224 clones examined from mixedly infected, homotypically immune mice. Twenty-nine reassortants (10.66%) were identified among 272 progeny clones from mixedly infected, heterotypically immune mice. Thus, reassortment was reduced more than 50-fold by homotypic immunity and approximately threefold by heterotypic immunity compared with prior data obtained from mixed infections of nonimmune mice. In addition, reassortment between SA11 and RRV in nonimmune mice was shown to be dependent on the virus dose. Taken together, these results suggest that immune responses may modulate the frequency of reassortment by reducing the effective multiplicity of infection (by neutralization or other immune mechanisms), thereby preventing efficient mixed infection of enterocytes.     

Monitoring in vivo changes in lung microstructure with ³He MRI in Sendai virus-infected mice

Recently, a Sendai virus (SeV) model of chronic obstructive lung disease has demonstrated an innate immune response in mouse airways that exhibits similarities to the chronic airway inflammation in human chronic obstructive pulmonary disease (COPD) and asthma, but the effect on distal lung parenchyma has not been investigated. The aim of our study is to image the time course and regional distribution of mouse lung microstructural changes in vivo after SeV infection. (1)H and (3)He diffusion magnetic resonance imaging (MRI) were successfully performed on five groups of C57BL/6J mice. (1)H MR images provided precise anatomical localization and lung volume measurements. (3)He lung morphometry was implemented to image and quantify mouse lung geometric microstructural parameters at different time points after SeV infection. (1)H MR images detected the SeV-induced pulmonary inflammation in vivo; spatially resolved maps of acinar airway radius R, alveolar depth h, and mean linear intercept Lm were generated from (3)He diffusion images. The morphometric parameters R and Lm in the infected group were indistinguishable from PBS-treated mice at day 21, increased slightly at day 49, and were increased with statistical significance at day 77 (p = 0.02). Increases in R and Lm of infected mice imply that there is a modest increase in alveolar duct radius distal to airway inflammation, particularly in the lung periphery, indicating airspace enlargement after virus infection. Our results indicate that (3)He lung morphometry has good sensitivity in quantifying small microstructural changes in the mouse lung and that the Sendai mouse model has the potential to be a valid murine model of COPD.     

Virus-neutralizing antibodies of immunoglobulin G (IgG) but not of IgM or IgA isotypes can cure influenza virus pneumonia in SCID mice.

The ability of monoclonal antibodies (MAbs) to passively cure an influenza virus pneumonia in the absence of endogenous T- and B-cell responses was investigated by treating C.B-17 mice, homozygous for the severe combined immunodeficiency (SCID) mutation, with individual monoclonal antiviral antibodies 1 day after pulmonary infection with influenza virus PR8 [A/PR/8/34 (H1N1)]. Less than 10% of untreated SCID mice survived the infection. By contrast, 100% of infected SCID mice that had been treated with a single intraperitoneal inoculation of at least 175 micrograms of a pool of virus-neutralizing (VN+) antihemagglutinin (anti-HA) MAbs survived, even if antibody treatment was delayed up to 7 days after infection. The use of individual MAbs showed that recovery could be achieved by VN+ anti-HA MAbs of the immunoglobulin G1 (IgG1), IgG2a, IgG2b, and IgG3 isotypes but not by VN+ anti-HA MAbs of the IgA and IgM isotypes, even if the latter were used in a chronic treatment protocol to compensate for their shorter half-lives in vivo. Both IgA and IgM, although ineffective therapeutically, protected against infection when given prophylactically, i.e., before exposure to virus. An Fc gamma-specific effector mechanism was not an absolute requirement for antibody-mediated recovery, as F(ab')2 preparations of IgGs could cure the disease, although with lesser efficacy, than intact IgG. An anti-M2 MAb of the IgG1 isotype, which was VN- but bound well to infected cells and inhibited virus growth in vitro, failed to cure. These observations are consistent with the idea that MAbs of the IgG isotype cure the disease by neutralizing all progeny virus until all productively infected host cells have died. VN+ MAbs of the IgA and IgM isotypes may be ineffective therapeutically because they do not have sufficient access to all tissue sites in which virus is produced during influenza virus pneumonia.     

Cellular Changes in Blood Indicate Severe Respiratory Disease during Influenza Infections in Mice

Influenza A infection is a serious threat to human and animal health. Many of the biological mechanisms of the host-pathogen-interactions are still not well understood and reliable biomarkers indicating the course of the disease are missing. The mouse is a valuable model system enabling us to study the local inflammatory host response and the influence on blood parameters under controlled circumstances. Here, we compared the lung and peripheral changes after PR8 (H1N1) influenza A virus infection in C57BL/6J and DBA/2J mice using virus variants of different pathogenicity resulting in non-lethal and lethal disease. We monitored hematological and immunological parameters revealing that the granulocyte to lymphocyte ratio in the blood represents an early indicator of severe disease progression already two days after influenza A infection in mice. These findings might be relevant to optimize early diagnostic options of severe influenza disease and to monitor successful therapeutic treatment in humans.     

Immune responses and viral replication in long-term inapparent carrier ponies inoculated with equine infectious anemia virus

Persistent infection of equids by equine infectious anemia virus (EIAV) is typically characterized by a progression during the first year postinfection from chronic disease with recurring disease cycles to a long-term asymptomatic infection that is maintained indefinitely. The goal of the current study was to perform a comprehensive longitudinal analysis of the course of virus infection and development of host immunity in experimentally infected horses as they progressed from chronic disease to long-term inapparent carriage. We previously described the evolution of EIAV genomic quasispecies (C. Leroux, C. J. Issel, and R. C. Montelaro, J. Virol. 71:9627-9639, 1997) and host immune responses (S. A. Hammond, S. J. Cook, D. L. Lichtenstein, C. J. Issel, and R. C. Montelaro, J. Virol. 71:3840-3852, 1997) in four experimentally infected ponies during sequential disease episodes associated with chronic disease during the first 10 months postinfection. In the current study, we extended the studies of these experimentally infected ponies to 3 years postinfection to characterize the levels of virus replication and development of host immune responses associated with the progression from chronic disease to long-term inapparent infection. The results of these studies revealed over a 10(3)-fold difference in the steady-state levels of plasma viral RNA detected during long-term inapparent infection that correlated with the severity of chronic disease, indicating different levels of control of virus replication during long-term inapparent infections. Detailed analyses of antibody and cellular immune responses in all four ponies over the 3-year course of infection revealed a similar evolution during the first year postinfection of robust humoral and cellular immunity that then remained relatively constant during long-term inapparent infection. These observations indicate that immune parameters that have previously been correlated with EIAV vaccine protection fail to provide reliable immune correlates of control of virus replication or clinical outcome in experimental infections. Thus, these data emphasize the differences between immunity to virus exposure and immune control of an established viral infection and further emphasize the need to develop and evaluate novel immunoassays to define reliable immune correlates to vaccine and infection immunity, respectively.     

Protection of Rabbits and Immunodeficient Mice against Lethal Poxvirus Infections by Human Monoclonal Antibodies

Smallpox (variola virus) is a bioweapon concern. Monkeypox is a growing zoonotic poxvirus threat. These problems have resulted in extensive efforts to develop potential therapeutics that can prevent or treat potentially lethal poxvirus infections in humans. Monoclonal antibodies (mAbs) against smallpox are a conservative approach to this problem, as the licensed human smallpox vaccine (vaccinia virus, VACV) primarily works on the basis of protective antibody responses against smallpox. Fully human mAbs (hmAbs) against vaccinia H3 (H3L) and B5 (B5R), targeting both the mature virion (MV) and extracellular enveloped virion (EV) forms, have been developed as potential therapeutics for use in humans. Post-exposure prophylaxis was assessed in both murine and rabbit animal models. Therapeutic efficacy of the mAbs was assessed in three good laboratory practices (GLP) studies examining severe combined immunodeficiency mice (SCID) given a lethal VACV infection. Pre-exposure combination hmAb therapy provided significantly better protection against disease and death than either single hmAb or vaccinia immune globulin (VIG). Post-exposure combination mAb therapy provided significant protection against disease and death, and appeared to fully cure the VACV infection in ≥50% of SCID mice. Therapeutic efficacy was then assessed in two rabbit studies examining post-exposure hmAb prophylaxis against rabbitpox (RPXV). In the first study, rabbits were infected with RPVX and then provided hmAbs at 48 hrs post-infection, or 1 hr and 72 hrs post-infection. Rabbits in both groups receiving hmAbs were 100% protected from death. In the second rabbitpox study, 100% of animal treated with combination hmAb therapy and 100% of animals treated with anti-B5 hmAb were protected. These findings suggest that combination hmAb treatment may be effective at controlling smallpox disease in immunocompetent or immunodeficient humans.