Experimental viral evolution reveals major histocompatibility complex polymorphisms as the primary host factors controlling pathogen adaptation and virulence.

Using an experimental evolution approach, we recently demonstrated that the mouse-specific pathogen Friend virus (FV) complex adapted to specific major histocompatibility complex (MHC) genotypes, which resulted in fitness tradeoffs when viruses were exposed to hosts possessing novel MHC polymorphisms. Here we report the analysis of patterns of pathogen adaptation and virulence evolution from viruses adapting to one of three hosts that differ across the entire genome (A/WySn, DBA/2J and BALB/c). We found that serial passage of FV complex through these mouse genotypes resulted in significant increases in pathogen fitness (156-fold) and virulence (11-fold). Adaptive responses by post-passage viruses also resulted in host-genotype-specific patterns of adaptation. To evaluate the relative importance of MHC versus non-MHC polymorphisms as factors influencing pathogen adaptation and virulence, we compared the magnitude of fitness tradeoffs incurred by post-passage viruses when infecting hosts possessing either novel MHC polymorphisms alone or hosts possessing novel MHC and non-MHC polymorphisms. MHC polymorphisms alone accounted for 71% and 83% of the total observed reductions in viral fitness and virulence in unfamiliar host genotypes, respectively. Strikingly, these data suggest that genetic polymorphisms within the MHC, a gene region representing only -0.1% of the genome, are major host factors influencing pathogen adaptation and virulence evolution.

Requirement for multiple lymphocyte subsets in protection by a live attenuated vaccine against retroviral infection.

Infection by live attenuated retroviruses provides excellent protection from challenge with pathogenic viruses in several animal models, but little is known about which immune effectors are necessary for protection. We examined this using adoptive transfer experiments in the Friend virus mouse model. Transfers of immune spleen cells into naive mice conferred complete protection, and transfers of purified lymphocyte subsets demonstrated that this effect required complex immune responses involving CD4+ and CD8+ T cells and also B cells. In addition, passive immunization experiments demonstrated that antibodies alone reduced virus loads but did not prevent infection. These findings may have implications for retroviral vaccine design in general.

Cellular and molecular mechanisms of vaccine-induced protection against retroviral infections.

More than 15 years after the discovery of human immunodeficiency virus (HIV), researchers are still struggling to design a protective AIDS vaccine. A remaining problem is a lack of basic knowledge about the immunological requirements for protection against retroviruses. Infection of macaque monkeys with simian immunodeficiency virus is still the best model for HIV vaccine research. However, in this model it remains difficult to determine protective immunological mechanisms because of limited numbers of experimental animals and their genetic heterogeneity. Thus, fundamental concepts in retroviral immunology have to be defined in other ways such as mouse models. This minireview summarizes new findings on cellular and molecular mechanisms in protection of mice against Friend murine retrovirus infection. It has been shown that complex immune responses, including B and T cell responses, are required for efficient protection in this model. Multiple viral antigens are necessary to elicit such broad immune reactivity. Efficacious vaccines must protect not only against acute disease, but also against the establishment of persistent infections or the host is at serious risk of virus reactivation. The minireview closes with a discussion on the relevance of findings from the mouse model on the design of a protective vaccine against HIV.

Unique biochemical properties of a mutant MHC class I molecule, H-2Ksm1.

This study describes serological and biochemical properties of a novel MHC class I molecule. The mutant H-2Ksm1 molecule was discovered in a mouse because of loss of reactivity of its peripheral blood lymphocytes to monoclonal antibodies. This mutation in the H-2Ks molecule is the first in vivo mutation described that has altered an amino acid residue (amino acid 107) distant from the regions generally considered to be peptide or TCR contacts. Cell surface expression of the mutant molecules remains high but the Arg107 to Trp substitution appears to alter the native protein conformation, markedly decreasing cell surface association with beta 2-microglobulin light chains and conferring a loss of recognition by Ks specific antibodies.

Transplantation effects of a unique major histocompatibility complex class I mutation.

This study describes a novel MHC class I mouse mutant that was discovered because of loss of reactivity of its cells to monoclonal antibodies. The mutation occurred in the H-2Ks molecule and is the first in vivo mutation described that has a single altered amino acid residue (amino acid 107) distant from the regions considered to be peptide or TCR contacts. Nevertheless, skin grafts from the mutant to the parent are rejected by CD8+ T-cells. In the reciprocal direction, the mutant shows partial tolerance to parental skin grafts, suggesting that the mutant is inefficient in selecting alloreactive T-cells specific for the wild-type Ks molecule.

Lymphocyte deficiencies increase susceptibility to friend virus-induced erythroleukemia in Fv-2 genetically resistant mice.

The study of genetic resistance to retroviral diseases provides insights into the mechanisms by which organisms overcome potentially lethal infections. Fv-2 resistance to Friend virus-induced erythroleukemia acts through nonimmunological mechanisms to prevent early virus spread, but it does not completely block infection. The current experiments were done to determine whether Fv-2 alone could provide resistance or whether immunological mechanisms were also required to bring infection under control. Fv-2-resistant mice that were CD4(+) T-cell deficient were able to restrict early virus replication and spread as well as normal Fv-2-resistant mice, but they could not maintain control and developed severe Friend virus-induced splenomegaly and erythroleukemia by 6 to 8 weeks postinfection. Mice deficient in CD8(+) T cells and, to a lesser extent, B cells were also susceptible to late Friend virus-induced disease. Thus, Fv-2 resistance does not independently prevent FV-induced erythroleukemia but works in concert with the immune system by limiting early infection long enough to allow virus-specific immunity time to develop and facilitate recovery.

Immunity to retroviral infection: the Friend virus model.

Friend virus infection of adult immunocompetent mice is a well established model for studying genetic resistance to infection by an immunosuppressive retrovirus. This paper reviews both the genetics of immune resistance and the types of immune responses required for recovery from infection. Specific major histocompatibility complex (MHC) class I and II alleles are necessary for recovery, as is a non-MHC gene, Rfv-3, which controls virus-specific antibody responses. In concordance with these genetic requirements are immunological requirements for cytotoxic T lymphocyte, T helper, and antibody responses, each of which provides essential nonoverlapping functions. The complexity of responses necessary for recovery from Friend virus infection has implications for both immunotherapies and vaccines. For example, it is shown that successful passive antibody therapy is dependent on MHC type because of the requirement for T cell responses. For vaccines, successful immunization requires priming of both T cell and B cell responses. In vivo depletion experiments demonstrate different requirements for CD8(+) T cells depending on the vaccine used. The implications of these studies for human retroviral diseases are discussed.

Different immunological requirements for protection against acute versus persistent Friend retrovirus infections.

The propensity of retroviruses to rapidly establish persistent infections poses a formidable problem in vaccination strategies. In the current study, we use a live attenuated vaccine to study protection against acute and persistent Friend virus infections in mice. Adoptive transfers of immune CD8(+) T cells combined with passive immunizations with virus-neutralizing antibodies increased protection against acute disease compared with either treatment alone, but there was no protection against the establishment of persistent infection. In addition, the protection against acute disease elicited by the combination treatment was dependent on endogenous CD4(+) T cells as no protection was achieved in CD4(+) T-cell-depleted mice. Quantitative studies showed that doubling the numbers of immune lymphocytes used in adoptive transfer experiments increased protection against acute disease depending on the type of lymphocyte subset used in the transfer. CD8(+) T cells were the most potent subset for the transfer of such protection. However, even high numbers of immune CD8(+) T cells gave no protection against the establishment of persistent infections. The data indicate that strengthening the numbers of specific immune cell subsets may have a beneficial effect on protection against acute disease, but protection from establishment of persistence requires complex immune responses involving multiple lymphocyte subsets.

Characterization of a live-attenuated retroviral vaccine demonstrates protection via immune mechanisms.

Live-attenuated retroviruses have been shown to be effective retroviral vaccines, but currently little is known regarding the mechanisms of protection. In the present studies, we used Friend virus as a model to analyze characteristics of a live-attenuated vaccine in protection against virus-induced disease. Highly susceptible mice were immunized with nonpathogenic Friend murine leukemia helper virus (F-MuLV), which replicates poorly in adult mice. Further attenuation of the vaccine virus was achieved by crossing the Fv-1 genetic resistance barrier. The minimum dose of vaccine virus required to protect 100% of the mice against challenge with pathogenic Friend virus complex was determined to be 10(3) focus-forming units of attenuated virus. Live vaccine virus was necessary for induction of immunity, since inactivated F-MuLV did not induce protection. To determine whether immune cells mediated protection, spleen cells from vaccinated donor mice were adoptively transferred into syngeneic recipients. The results indicated that immune mechanisms rather than viral interference mediated protection.

Critical role for CD4(+) T cells in controlling retrovirus replication and spread in persistently infected mice.

Reactivations of persistent viral infections pose a significant medical problem in immunocompromised cancer, transplant, and AIDS patients, yet little is known about how persistent viral infections are immunologically controlled. Here we describe a mouse model for investigating the role of the immune response in controlling a persistent retroviral infection. We demonstrate that, following recovery from acute Friend virus infection, a small number of B cells evade immunological destruction and harbor persistent virus. In vivo depletions of T-cell subsets in persistently infected mice revealed a critical role for CD4(+) T cells in controlling virus replication, spread to the erythroid lineage, and induction of erythroleukemia. The CD4(+) T-cell effect was independent of CD8(+) T cells and in some cases was also independent of virus-neutralizing antibody responses. Thus, the CD4(+) T cells may have had a direct antiviral effect. These results may have relevance for human immunodeficiency virus (HIV) infections where loss of CD4(+) T cells is associated with an increase in HIV replication, reactivation of persistent viruses, and a high incidence of virus-associated cancers.

Passive immunotherapy for retroviral disease: influence of major histocompatibility complex type and T-cell responsiveness.

Administration of virus-specific antibodies is known to be an effective early treatment for some viral infections. Such immunotherapy probably acts by antibody-mediated neutralization of viral infectivity and is often thought to function independently of T-cell-mediated immune responses. In the present experiments, we studied passive antibody therapy using Friend murine leukemia virus complex as a model for an immunosuppressive retroviral disease in adult mice. The results showed that antibody therapy could induce recovery from a well-established retroviral infection. However, the success of therapy was dependent on the presence of both CD4+ and CD8+ T lymphocytes. Thus, cell-mediated responses were required for recovery from infection even in the presence of therapeutic levels of antibody. The major histocompatibility type of the mice was also an important factor determining the relative success of antibody therapy in this system, but it was less critical for low-dose than for high-dose infections. Our results imply that limited T-cell responsiveness as dictated by major histocompatibility genes and/or stage of disease may have contributed to previous immunotherapy failures in AIDS patients. Possible strategies to improve the efficacy of future therapies are discussed.

Kinetics of the development of protective immunity in mice vaccinated with a live attenuated retrovirus.

Vaccination of mice with a live attenuated vaccine virus induces potent protection against subsequent challenge with pathogenic Friend retroviral complex. The kinetic studies presented here demonstrate protection from acute splenomegaly as early as 1 week postvaccination. At this time point virus-specific cytotoxic T lymphocytes (CTL) were demonstrable in direct chromium release assays. However, during the first 2 weeks after vaccination protection was incomplete since the mice were not protected against establishment of low-level persistent infections in the spleen. By 3 weeks postvaccination the animals were protected against the establishment of persistent virus as well as acute splenomegaly. The timing of this complete protection correlated with the presence of both virus-neutralizing antibodies and primed CTL in the immunized mice. Within 3 days of virus challenge, vaccinated mice showed high levels of activated B cells and CD4(+) and CD8(+) T cells, indicating an efficient priming of all lymphocyte subsets. Despite very limited replication of the vaccine virus, the protective effect was long lived and was still present 6 months after immunization.

Protection against establishment of retroviral persistence by vaccination with a live attenuated virus.

Many human viruses not only cause acute diseases but also establish persistent infections. Such persistent viruses can cause chronic diseases or can reactivate to cause acute diseases in AIDS patients or patients receiving immunosuppressive therapies. While the prevention of persistent infections is an important consideration in the design of modern vaccines, surprisingly little is known about this aspect of protection. In the current study, we tested the feasibility of vaccine prevention of retroviral persistence by using a Friend virus model that we recently developed. In this model, persistent virus can be detected at very low levels by immunosuppressing the host to reactivate virus or by transferring persistently infected spleen cells into highly susceptible mice. Two vaccines were analyzed, a recombinant vaccinia virus vector expressing Friend virus envelope protein and a live attenuated Friend virus. Both vaccines reduced pathogenic virus loads to levels undetectable by infectious center assays. However, only the live, attenuated vaccine prevented immunosuppression-induced reactivation of persistent virus. Thus, even very low levels of persistent Friend virus posed a significant threat during immunosuppression. Our results demonstrate that vaccine protection against establishment of retroviral persistence is attainable.

CD4(+) T cells and gamma interferon in the long-term control of persistent friend retrovirus infection.

We have used the Friend virus model to determine the basic mechanisms by which the immune system can control persistent retroviral infections. Previously we showed that CD4(+) T cells play an essential role in keeping persistent retrovirus in check. The present in vitro experiments with a Friend virus-specific CD4(+) T-cell clone revealed that these cells produce gamma interferon (IFN-gamma), which acts with two distinct mechanisms of antiviral activity. First, IFN-gamma had a direct inhibitory effect on virus production. This inhibitory effect was noncytolytic and, interestingly, was not associated with decreased cell surface expression of viral antigens. The second mechanism of IFN-gamma-mediated antiviral activity was an enhancement of CD4(+) T-cell-mediated cytolytic activity. We also found an in vivo role for IFN-gamma in the control of persistent Friend virus infections. Neutralization of IFN-gamma in persistently infected mice resulted in significantly increased levels of virus in the spleen, and a significant percentage of IFN-gamma-deficient mice were unable to maintain long-term control over Friend virus infections.

Neurologic disease induced by polytropic murine retroviruses: neurovirulence determined by efficiency of spread to microglial cells.

Several murine leukemia viruses (MuLV) induce neurologic disease in susceptible mice. To identify features of central nervous system (CNS) infection that correlate with neurovirulence, we compared two neurovirulent MuLV, Fr98 and Fr98/SE, with a nonneurovirulent MuLV, Fr54. All three viruses utilize the polytropic receptor and are coisogenic, each containing a different envelope gene within a common genetic background. Both Fr98 and Fr98/SE induce a clinical neurologic disease characterized by hyperexcitability and ataxia yet differ in incubation period, 16 to 30 and 30 to 60 days, respectively. Fr54 infects the CNS but fails to induce clinical signs of neurologic disease. In this study, we compared the histopathology, regional virus distribution, and cell tropism in the brain, as well as the relative CNS viral burdens. All three viruses induced similar histopathologic effects, characterized by intense reactive astrogliosis and microglial activation associated with minimal vacuolar degeneration. The infected target cells for each virus consisted primarily of endothelial and microglial cells, with rare oligodendrocytes. Infection localized predominantly in white matter tracts of the cerebellum, internal capsule, and corpus callosum. The only feature that correlated with relative neurovirulence was viral burden as measured by both viral CA protein expression in cerebellar homogenates and quantification of infected cells. Interestingly, Fr54 (nonneurovirulent) and Fr98/SE (slow disease) had similar viral burdens at 3 weeks postinoculation, suggesting that they entered the brain with comparable efficiencies. However, spread of Fr98/SE within the brain thereafter exceeded that of Fr54, reaching levels of viral burden comparable to that seen for Fr98 (rapid disease) at 3 weeks. These results suggest that the determinants of neurovirulence in the envelope gene may influence the efficiency of virus spread within the brain and that a critical number of infected cells may be required for induction of clinical neurologic disease.

Two separate envelope regions influence induction of brain disease by a polytropic murine retrovirus (FMCF98).

The major determinants involved in neurological disease induction by polytropic murine leukemia virus FMCF98 are encoded by the envelope gene. To map these determinants further, we produced four chimeras which contained neurovirulent FMCF98 envelope sequences combined with envelope sequences from the closely related nonneurovirulent polytropic virus FMCF54. Surprisingly, two chimeric viruses containing completely separate envelope regions from FMCF98 could both induce neurological disease. Clinical signs caused by both neurovirulent chimeras appeared to be indistinguishable from those caused by FMCF98, although the incubation periods were longer. One neurovirulence determinant mapped to the N-terminal portion of gp7O, which contains the VRA and VRB receptor-binding regions, while the other determinant mapped downstream of both of the variable regions. Western blot (immunoblot) analyses and immunohistochemical staining of tissue sections indicated that the variations in neurovirulence of these viruses could not be explained by differences in either the quantitative level or the location of virus expression in the brain.

Recovery from Friend disease in mice with reduced major histocompatibility complex class I expression.

Mice homozygous for the b allele of the MHC gene, H-2D, have a high incidence of recovery from Friend virus infections, while mice heterozygous for the b allele at H-2D have a very low incidence of recovery. Previous experiments indicated that the low recovery rates associated with heterozygosity at H-2D might be related to a gene dosage effect requiring the expression of two H-2Db alleles for high recovery. We investigated the effects of reduced H-2Db expression on recovery from Friend disease by using H-2b homozygous mice carrying a single beta 2-microglobulin gene disruption. These mice had reductions in cell surface H-2Db expression comparable to those of H-2Da/b heterozygotes. Numerous cell types with various levels of H-2Db expression were examined, and in each case, the expression levels in the beta 2-microglobulin mutants closely reflected those observed in the H-2Da/b heterozygotes. We found, however, that reduced expression did not affect recovery from Friend disease, indicating that heterozygous levels of H-2Db expression are sufficient for the high-recovery phenotype previously associated only with H-2Db homozygotes.

Immunoprotective determinants in friend murine leukemia virus envelope protein.

Several immunological epitopes are known to be located within the Friend murine leukemia virus (F-MuLV) envelope protein, but their relative contributions to protection from Friend virus-induced disease are not known. To determine how expression of various immunological determinants affected protection, mice were immunized with recombinant vaccinia viruses expressing different portions of the F-MuLV envelope protein, and they were then challenged with a lethal dose of Friend virus complex. The disease parameters that were followed in the mice were early viremia, early splenomegaly, and late splenomegaly. Both the N-terminal and C-terminal portions of the F-MuLV gp70 were found to protect against late splenomegaly, the primary clinical sign associated with virus-induced erythroleukemia. However, neither region alone protected against early splenomegaly and early viremia, indicating poor immunological control over early virus replication and spread through the spleen and blood. In contrast, mice immunized with a vaccine expressing the entire F-MuLV envelope protein were protected against all three disease parameters. The results indicated that expression of multiple immunological determinants including both T-helper and B cell epitopes was necessary for full protection.

Major histocompatibility complex class I gene controls the generation of gamma interferon-producing CD4(+) and CD8(+) T cells important for recovery from friend retrovirus-induced leukemia.

Recovery from leukemia induced by Friend virus complex (FV) requires strong CD4(+) helper, CD8(+) cytotoxic T-lymphocyte, and B-cell responses. The development of these immune responses is dependent on the major histocompatibility complex (MHC) (H-2) genotype of the mouse. In H-2(b/b) mice, which spontaneously recover from FV-induced erythroleukemia, neutralization of gamma interferon (IFN-gamma) in vivo inhibited recovery, which indicated that IFN-gamma was a necessary component of the immune response to FV. Furthermore, in H-2(b/b) mice, high numbers of IFN-gamma-producing cells were detected after FV infection, whereas in H-2(a/b) mice, which have a low-recovery phenotype, only low numbers of IFN-gamma-producing cells were detected. Similarly, H-2(bm14/b) mice, which cannot recover from FV infection due to a point mutation in one allele of the H-2D(b) gene, also had low numbers of IFN-gamma-producing T cells. Surprisingly, this effect was observed for both CD8(+) and CD4(+) T cells. These findings reveal a novel influence of MHC class I genes on CD4(+) T-cell responses to viral infection. Furthermore, the influence of MHC class I genotype on the generation of both IFN-gamma-producing CD4(+) and CD8(+) T cells helps explain the major impact of the H-2D gene on recovery from FV disease.