Immunization with a murine cytomegalovirus based vector encoding retrovirus envelope confers strong protection from Friend retrovirus challenge infection.

Immunization vectors based on cytomegalovirus (CMV) have attracted a lot of interest in recent years because of their high efficacy in the simian immunodeficiency virus (SIV) macaque model, which has been attributed to their ability to induce strong, unusually broad, and unconventionally restricted CD8+ T cell responses. To evaluate the ability of CMV-based vectors to mediate protection by other immune mechanisms, we evaluated a mouse CMV (MCMV)-based vector encoding Friend virus (FV) envelope (Env), which lacks any known CD8+ T cell epitopes, for its protective efficacy in the FV mouse model. When we immunized highly FV-susceptible mice with the Env-encoding MCMV vector (MCMV.env), we could detect high frequencies of Env-specific CD4+ T cells after a single immunization. While the control of an early FV challenge infection was highly variable, an FV infection applied later after immunization was tightly controlled by almost all immunized mice. Protection of mice correlated with their ability to mount a robust anamnestic neutralizing antibody response upon FV infection, but Env-specific CD4+ T cells also produced appreciable levels of interferon γ. Depletion and transfer experiments underlined the important role of antibodies for control of FV infection but also showed that while no Env-specific CD8+ T cells were induced by the MCMV.env vaccine, the presence of CD8+ T cells at the time of FV challenge was required. The immunity induced by MCMV.env immunization was long-lasting, but was restricted to MCMV naïve animals. Taken together, our results demonstrate a novel mode of action of a CMV-based vaccine for anti-retrovirus immunization that confers strong protection from retrovirus challenge, which is conferred by CD4+ T cells and antibodies.

Experimental manipulation of population-level MHC diversity controls pathogen virulence evolution in Mus musculus.

The virulence levels attained by serial passage of pathogens through similar host genotypes are much higher than observed in natural systems; however, it is unknown what keeps natural virulence levels below these empirically demonstrated maximum levels. One hypothesis suggests that host diversity impedes pathogen virulence, because adaptation to one host genotype carries trade-offs in the ability to replicate and cause disease in other host genotypes. To test this hypothesis, with the simplest level of population diversity within the loci of the major histocompatibility complex (MHC), we serially passaged Friend virus complex (FVC) through two rounds, in hosts with either the same MHC genotypes (pure passage) or hosts with different MHC genotypes (alternated passage). Alternated passages showed a significant overall reduction in viral titre (31%) and virulence (54%) when compared to pure passages. Furthermore, a resistant host genotype initially dominated any effects due to MHC diversity; however, when FVC was allowed to adapt to the resistant host genotype, predicted MHC effects emerged; that is, alternated lines show reduced virulence. These data indicate serial exposure to diverse MHC genotypes is an impediment to pathogen adaptation, suggesting genetic variation at MHC loci is important for limiting virulence in a rapidly evolving pathogen and supports negative frequency-dependent selection as a force maintaining MHC diversity in host populations.

Mutation of the Putative Immunosuppressive Domain of the Retroviral Envelope Glycoprotein Compromises Infectivity.

The envelope glycoprotein of diverse endogenous and exogenous retroviruses is considered inherently immunosuppressive. Extensive work mapped the immunosuppressive activity to a highly conserved domain, termed the immunosuppressive domain (ISD), in the transmembrane (TM) subunit of the envelope glycoprotein and identified two naturally polymorphic key residues that afford immunosuppressive activity to distinct envelope glycoproteins. Concurrent mutation of these two key residues (E14R and A20F) in the envelope glycoprotein of the Friend murine leukemia virus (F-MLV) ISD has been reported to abolish its immunosuppressive activity, without affecting its fusogenicity, and to weaken the ability of the virus to replicate specifically in immunocompetent hosts. Here, we show that mutation of these key residues did, in fact, result in a substantial loss of F-MLV infectivity, independently of host immunity, challenging whether associations exist between the two. Notably, a loss of infectivity incurred by the F-MLV mutant with the E14R and A20F double ISD mutation was conditional on expression of the ecotropic envelope receptor murine cationic amino acid transporter-1 (mCAT1) in the virus-producing cell. Indeed, the F-MLV mutant retained infectivity when it was produced by human cells, which naturally lack mCAT1 expression, but not by murine cells. Furthermore, mCAT1 overexpression in human cells impaired the infectivity of both the F-MLV double mutant and the wild-type F-MLV strain, suggesting a finely tuned relationship between the levels of mCAT1 in the producer cell and the infectivity of the virions produced. An adverse effect on this relationship, rather than disruption of the putative ISD, is therefore more likely to explain the loss of F-MLV infectivity incurred by mutations in key ISD residues E14 and A20.IMPORTANCE Retroviruses can interact with their hosts in ways that, although not entirely understood, can greatly influence their pathogenic potential. One such example is a putative immunosuppressive activity, which has been mapped to a conserved domain of the retroviral envelope glycoprotein of several exogenous as well as endogenous retroviruses. In this study, mutations naturally found in some envelope glycoproteins lacking immunosuppressive activity were shown to affect retrovirus infectivity only if the host cell that produced the retrovirus also expressed the cellular entry receptor. These findings shed light on a novel role for this conserved domain in providing the necessary stability to the envelope glycoprotein in order to withstand the interaction with the cellular receptor during virus formation. This function of the domain is critical for further elucidation of the mechanism of immunosuppression mediated by the retroviral envelope glycoprotein.

Fli-1 overexpression in erythroleukemic cells promotes erythroid de-differentiation while Spi-1/PU.1 exerts the opposite effect.

The ETS transcription factors play a critical role during hematopoiesis. In F-MuLV-induced erythroleukemia, Fli­1 insertional activation producing high expression of this transcription factor required to promote proliferation. How deregulated Fli­1 expression alters the balance between erythroid differentiation and proliferation is unknown. To address this issue, we exogenously overexpressed Fli­1 in an erythroleukemic cell harboring activation of spi­1/PU.1, another ETS gene involved in erythroleukemogenesis. While the proliferation in culture remains unaffected, Fli­1 overexpression imparts morphological and immunohistochemical characteristics of immature erythroid progenitors. Fli­1 overexpression in erythroleukemic cells increased the numbers of erythroid colonies on methylcellulose and reduced tumorigenicity as evidenced by increase latency of erythroleukemogenesis in mice inoculated with these cells. Although all transplanted mice developed enlargement of the spleen and liver due to leukemic infiltration, Fli­1 overexpression altered the hematopoietic phenotype, significantly increasing the expression of regulatory hematopoietic genes cKIT, SCA-1, CD41 and CD71. In contrast, expression of Spi­1/PU.1 in a Fli­1 producing erythroleukemia cell line in which fli­1 is activated, resulted in increased proliferation through activation of growth promoting proteins MAPK, AKT, cMYC and JAK2. Importantly, these progenitors express high levels of markers such as CD71 and TER119 associated with more mature erythroid cells. Thus, Fli­1 overexpression induces a de-differentiation program by reverting CFU-E to BFU-E erythroid progenitor activity, while Spi­1/PU.1 promoting maturation from BFU-E to CFU-E.

Reduced Competitive Repopulation Capacity of Multipotential Hematopoietic Stem Cells in the Bone Marrow of Friend Virus-infected Fv2-resistant Mice.

BACKGROUND/AIM: The polycythemia form of Friend leukemia virus (FVP) causes splenomegaly and lethal erythroleukemia in Fv-2s-susceptible mouse strains. We sought to determine whether the hematopoietic stem cell (HSC) pool was expanded in Fv-2r-resistant mice. MATERIALS AND METHODS: The 120-day bone marrow transplantation competitive repopulation assay was used to determine whether FVP-infected Fv-2r C57BL/6 mice demonstrated expansion of the HSC pool compared to the pool of committed hematopoietic progenitor cells in the same marrow assayed in vitro. RESULTS: There was a significant expansion of committed hematopoietic progenitors observed in virus-infected Fv-2s FVB mice, but not Fv-2r C57BL/6 mice. Furthermore, Fv-2r mice showed no detectable expansion of either committed hematopoietic progenitor cells or the multipotential stem cell pool by competitive repopulation assay. CONCLUSION: Friend virus disease in Fv-2s mice is associated with expansion of committed hematopoietic progenitors. Fv-2r mice show no expansion of either committed progenitor or pluripotential stem cell numbers.

TLR ligand induced IL-6 counter-regulates the anti-viral CD8(+) T cell response during an acute retrovirus infection.

We have previously shown that Toll-like receptor (TLR) agonists contribute to the control of viral infection by augmenting virus-specific CD8(+) T-cell responses. It is also well established that signaling by TLRs results in the production of pro-inflammatory cytokines such as interleukin 6 (IL-6). However, how these pro-inflammatory cytokines influence the virus-specific CD8(+) T-cell response during the TLR agonist stimulation remained largely unknown. Here, we investigated the role of TLR-induced IL-6 in shaping virus-specific CD8(+) T-cell responses in the Friend retrovirus (FV) mouse model. We show that the TLR agonist induced IL-6 counter-regulates effector CD8(+) T-cell responses. IL-6 potently inhibited activation and cytokine production of CD8(+) T cells in vitro. This effect was mediated by a direct stimulation of CD8(+) T cells by IL-6, which induced upregulation of STAT3 phosphorylation and SOCS3 and downregulated STAT4 phosphorylation and T-bet. Moreover, combining TLR stimulation and IL-6 blockade during an acute FV infection resulted in enhanced virus-specific CD8(+) T-cell immunity and better control of viral replication. These results have implications for our understanding of the role of TLR induced pro-inflammatory cytokines in regulating effector T cell responses and for the development of therapeutic strategies to overcome T cell dysfunction in chronic viral infections.

Serial infection of diverse host (Mus) genotypes rapidly impedes pathogen fitness and virulence.

Reduced genetic variation among hosts may favour the emergence of virulent infectious diseases by enhancing pathogen replication and its associated virulence due to adaptation to a limited set of host genotypes. Here, we test this hypothesis using experimental evolution of a mouse-specific retroviral pathogen, Friend virus (FV) complex. We demonstrate rapid fitness (i.e. viral titre) and virulence increases when FV complex serially infects a series of inbred mice representing the same genotype, but not when infecting a diverse array of inbred mouse strains modelling the diversity in natural host populations. Additionally, a single infection of a different host genotype was sufficient to constrain the emergence of a high fitness/high virulence FV complex phenotype in these experiments. The potent inhibition of viral fitness and virulence was associated with an observed loss of the defective retroviral genome (spleen focus-forming virus), whose presence exacerbates infection and drives disease in susceptible mice. Results from our experiments provide an important first step in understanding how genetic variation among vertebrate hosts influences pathogen evolution and suggests that serial exposure to different genotypes within a single host species may act as a constraint on pathogen adaptation that prohibits the emergence of more virulent infections. From a practical perspective, these results have implications for low-diversity host populations such as endangered species and domestic animals.

Insertional activation of myb by F-MuLV in SCID mice induces myeloid leukemia.

Identification of retrovirus integration sites is a powerful method to identify cancer-related genes. This approach led to the discovery of the Friend murine leukemia virus (F-MuLV) integration site-1 (fli-1). Viral insertion at the fli-1 locus induces erythroleukemia in susceptible strains of mice. Our recent data demonstrated that, F-MuLV-infected SCID mice, in contrast to wt CB17 controls, developed a non­erythroleukemic leukemia without viral integration at the fli-1 locus. Using ligation-mediated polymerase chain reaction (LM-PCR) approach we identified a total of 15 viral integration sites in F-MuLV-infected SCID mice. One of the identified insertion sites was located about 62 kb upstream of the myeloblastosis (myb) gene. While integration within or surrounding the myb gene has been reported before for murine leukemia viruses, the location of the viral integration site identified in F-MuLV­infected SCID mice is novel and has never been reported. Using PCR analysis we showed that viral integration at the myb locus occurs with a frequency of 35% and therefore is considered as a common integration site. Integration of F-MuLV in this locus resulted in upregulation of the MYB protein. Flow cytometry analysis and methylcellulose culture of leukemic cells isolated from tumors with viral integration close to the myb indicated tumors of myeloid origin. Our findings indicate that, in contrast to wt CB17 mice, F-MuLV-infected SCID mice display viral integration within myeloid specific gene loci that result in the development of myelogenous leukemia.

Host resistance influences patterns of experimental viral adaptation and virulence evolution.

Infectious diseases are major threats to all living systems, so understanding the forces of selection that limit the evolution of more virulent pathogens is of fundamental importance; this includes the practical application of identifying possible mitigation strategies for at-risk host populations. The evolution of more virulent pathogens has been classically understood to be limited by the tradeoff between within-host growth rate and transmissibility. Importantly, heterogeneity among hosts can influence both of these factors. However, despite our substantial understanding of how the immune system operates to control pathogen replication during infection, we have only a limited appreciation of how variability in intrinsic (i.e., genetically determined) levels of host resistance influences patterns of pathogen adaptation and virulence evolution. Here, we describe results from experimental evolution studies using a model host-pathogen (virus-mammal) system; we demonstrate that variability in intrinsic levels of resistance among host genotypes can have significant effects on patterns of pathogen adaptation and virulence evolution during serial passage. Both the magnitude of adaptive response as well as the degree of pathogen specialization was positively correlated with host resistance, while mean overall virulence of post-passage virus was negatively correlated with host resistance. These results are consistent with a model whereby resistant host genotypes impose stronger selection on adapting pathogen populations, which in turn leads to the evolution of more specialized pathogen variants whose overall (i.e., mean) virulence across host genotypes is reduced.

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.

IFN-α treatment inhibits acute Friend retrovirus replication primarily through the antiviral effector molecule Apobec3.

Therapeutic administration of IFN-α in clinical trials significantly reduced HIV-1 plasma viral load and human T-lymphotropic virus type I proviral load in infected patients. The mechanism may involve the concerted action of multiple antiretroviral effectors collectively known as "restriction factors," which could vary in relative importance according to the magnitude of transcriptional induction. However, direct genetic approaches to identify the relevant IFN-α restriction factors will not be feasible in humans in vivo. Meanwhile, mice encode an analogous set of restriction factor genes and could be used to obtain insights on how IFN-α could inhibit retroviruses in vivo. As expected, IFN-α treatment of mice significantly upregulated the transcription of multiple restriction factors including Tetherin/BST2, SAMHD1, Viperin, ISG15, OAS1, and IFITM3. However, a dominant antiretroviral factor, Apobec3, was only minimally induced. To determine whether Apobec3 was necessary for direct IFN-α antiretroviral action in vivo, wild-type and Apobec3-deficient mice were infected with Friend retrovirus, then treated with IFN-α. Treatment of infected wild-type mice with IFN-α significantly reduced acute plasma viral load 28-fold, splenic proviral load 5-fold, bone marrow proviral load 14-fold, and infected bone marrow cells 7-fold, but no inhibition was observed in Apobec3-deficient mice. These findings reveal that IFN-α inhibits acute Friend retrovirus infection primarily through the antiviral effector Apobec3 in vivo, demonstrate that transcriptional induction levels did not predict the mechanism of IFN-α-mediated control, and highlight the potential of the human APOBEC3 proteins as therapeutic targets against pathogenic retrovirus infections.

Negative impact of IFN-γ on early host immune responses to retroviral infection.

The immune system is tasked with defending against a myriad of microbial infections, and its response to a given infectious microbe may be strongly influenced by coinfection with another microbe. It was shown that infection of mice with lactate dehydrogenase-elevating virus (LDV) impairs early adaptive immune responses to Friend virus (FV) coinfection. To investigate the mechanism of this impairment, we examined LDV-induced innate immune responses and found LDV-specific induction of IFN-α and IFN-γ. LDV-induced IFN-α had little effect on FV infection or immune responses, but unexpectedly, LDV-induced IFN-γ production dampened Th1 adaptive immune responses and enhanced FV infection. Two distinct effects were identified. First, LDV-induced IFN-γ signaling indirectly modulated FV-specific CD8+ T cell responses. Second, intrinsic IFN-γ signaling in B cells promoted polyclonal B cell activation and enhanced early FV infection, despite promotion of germinal center formation and neutralizing Ab production. Results from this model reveal that IFN-γ production can have detrimental effects on early adaptive immune responses and virus control.

Experimental viral evolution to specific host MHC genotypes reveals fitness and virulence trade-offs in alternative MHC types.

The unprecedented genetic diversity found at vertebrate MHC (major histocompatibility complex) loci influences susceptibility to most infectious and autoimmune diseases. The evolutionary explanation for how these polymorphisms are maintained has been controversial. One leading explanation, antagonistic coevolution (also known as the Red Queen), postulates a never-ending molecular arms race where pathogens evolve to evade immune recognition by common MHC alleles, which in turn provides a selective advantage to hosts carrying rare MHC alleles. This cyclical process leads to negative frequency-dependent selection and promotes MHC diversity if two conditions are met: (i) pathogen adaptation must produce trade-offs that result in pathogen fitness being higher in familiar (i.e., host MHC genotype adapted to) vs. unfamiliar host MHC genotypes; and (ii) this adaptation must produce correlated patterns of virulence (i.e., disease severity). Here we test these fundamental assumptions using an experimental evolutionary approach (serial passage). We demonstrate rapid adaptation and virulence evolution of a mouse-specific retrovirus to its mammalian host across multiple MHC genotypes. Critically, this adaptive response results in trade-offs (i.e., antagonistic pleiotropy) between host MHC genotypes; both viral fitness and virulence is substantially higher in familiar versus unfamiliar MHC genotypes. These data are unique in experimentally confirming the requisite conditions of the antagonistic coevolution model of MHC evolution and providing quantification of fitness effects for pathogen and host. These data help explain the unprecedented diversity of MHC genes, including how disease-causing alleles are maintained.

Noninfectious retrovirus particles drive the APOBEC3/Rfv3 dependent neutralizing antibody response.

Members of the APOBEC3 family of deoxycytidine deaminases counteract a broad range of retroviruses in vitro through an indirect mechanism that requires virion incorporation and inhibition of reverse transcription and/or hypermutation of minus strand transcripts in the next target cell. The selective advantage to the host of this indirect restriction mechanism remains unclear, but valuable insights may be gained by studying APOBEC3 function in vivo. Apobec3 was previously shown to encode Rfv3, a classical resistance gene that controls the recovery of mice from pathogenic Friend retrovirus (FV) infection by promoting a more potent neutralizing antibody (NAb) response. The underlying mechanism does not involve a direct effect of Apobec3 on B cell function. Here we show that while Apobec3 decreased titers of infectious virus during acute FV infection, plasma viral RNA loads were maintained, indicating substantial release of noninfectious particles in vivo. The lack of plasma virion infectivity was associated with a significant post-entry block during early reverse transcription rather than G-to-A hypermutation. The Apobec3-dependent NAb response correlated with IgG binding titers against native, but not detergent-lysed virions. These findings indicate that innate Apobec3 restriction promotes NAb responses by maintaining high concentrations of virions with native B cell epitopes, but in the context of low virion infectivity. Finally, Apobec3 restriction was found to be saturable in vivo, since increasing FV inoculum doses resulted in decreased Apobec3 inhibition. By analogy, maximizing the release of noninfectious particles by modulating APOBEC3 expression may improve humoral immunity against pathogenic human retroviral infections.

Toll-like receptor 7 controls the anti-retroviral germinal center response.

The development of vaccines that can enhance immunity to viral pathogens is an important goal. However, the innate molecular pathways that regulate the strength and quality of the immune response remain largely uncharacterized. To define the role of Toll-like receptor (TLR) signaling in control of a model retroviral pathogen, Friend virus (FV), I generated mice in which the TLR signaling adapter Myd88 was selectively deleted in dendritic cell (DC) or in B cell lineages. Deletion of Myd88 in DCs had little effect on immune control of FV, while B cell specific deletion of Myd88 caused a dramatic increase in viral infectious centers and a significantly reduced antibody response, indicating that B cell-intrinsic TLR signaling plays a crucial role, while TLR signaling in DCs is less important. I then identified the single-stranded RNA sensing protein TLR7 as being required for antibody-mediated control of FV by analyzing mice deficient in TLR7. Remarkably, B cells in infected TLR7-deficient mice upregulated CD69 and CD86 early in infection, but failed to develop into germinal center B cells. CD4 T cell responses were also attenuated in the absence of TLR7, but CD8 responses were TLR7 independent, suggesting the existence of additional pathways for detection of retroviral particles. Together these results demonstrate that the vertebrate immune system detects retroviruses in vivo via TLR7 and that this pathway regulates a key checkpoint controlling development of germinal center B cells.

Narrowing down the critical region within env gene for determining neuropathogenicity of murine leukemia virus A8.

Friend murine leukemia virus clone A8 causes spongiform neurodegeneration in the rat brain, and the env gene of A8 is a primary determinant of neuropathogenicity. In order to narrow down the critical region within the env gene that determines neuropathogenicity, we constructed chimeric viruses having chimeric env between A8 and non-neuropathogenic 57 on the background of A8 virus. After replacement of the BamHI (at nucleotide 5715)-AgeI (at nucleotide 6322) fragment of A8 virus with the corresponding fragment of 57, neuropathogenicity was lost. In contrast, the chimeric viruses that have the BamHI (5715)-AgeI (6322) fragment of A8 induced spongiosis in 100% of infected rats at the same or slightly lower intensity than A8 virus. These results indicate that the BamHI (5715)-AgeI (6322) fragment of A8, which contains the signal sequence and the N-terminal half of RBD, is crucial for the induction of spongiform neurodegeneration. In the BamHI (5715)-AgeI (6322) fragment, seven amino acids differed between A8 and 57, one in the signal sequence and six in RBD, which suggests that these amino acids significantly contribute to the neuropathogenicity of A8.

Persistent Friend virus replication and disease in Apobec3-deficient mice expressing functional B-cell-activating factor receptor.

Rfv3 is an autosomal dominant gene that influences the recovery of resistant mice from Friend retrovirus (FV) infection by limiting viremia and promoting a more potent neutralizing antibody response. We previously reported that Rfv3 is encoded by Apobec3, an innate retrovirus restriction factor. However, it was recently suggested that the Rfv3 susceptible phenotype of high viremia at 28 days postinfection (dpi) was more dominantly controlled by the B-cell-activating factor receptor (BAFF-R), a gene that is linked to but located outside the genetically mapped region containing Rfv3. Although one prototypical Rfv3 susceptible mouse strain, A/WySn, indeed contains a dysfunctional BAFF-R, two other Rfv3 susceptible strains, BALB/c and A.BY, express functional BAFF-R genes, determined on the basis of genotyping and B-cell immunophenotyping. Furthermore, transcomplementation studies in (C57BL/6 [B6] × BALB/c)F(1) and (B6 × A.BY)F(1) mice revealed that the B6 Apobec3 gene significantly influences recovery from FV viremia, cellular infection, and disease at 28 dpi. Finally, the Rfv3 phenotypes of prototypic B6, A.BY, A/WySn, and BALB/c mouse strains correlate with reported Apobec3 mRNA expression levels. Overall, these findings argue against the generality of BAFF-R polymorphisms as a dominant mechanism to explain the Rfv3 recovery phenotype and further strengthen the evidence that Apobec3 encodes Rfv3.

Complement opsonization enhances friend virus infection of B cells and thereby amplifies the virus-specific CD8+ T cell response.

B cells are one of the targets of Friend virus (FV) infection, a well-established mouse model often used to study retroviral infections in vivo. Although B cells may be effective in stimulating cytotoxic T lymphocyte responses, studies involving their role in FV infection have mainly focused on neutralizing antibody production. Here we show that polyclonal activation of B cells promotes their infection with FV both in vitro and in vivo. Furthermore, we demonstrate that complement opsonization of Friend murine leukemia virus (F-MuLV) enhances infection of B cells, which correlates with increased potency of B cells to activate FV-specific CD8(+) T cells.

Long-lasting protective antiviral immunity induced by passive immunotherapies requires both neutralizing and effector functions of the administered monoclonal antibody.

Using FrCas(E) retrovirus-infected newborn mice as a model system, we have shown recently that a long-lasting antiviral immune response essential for healthy survival emerges after a short treatment with a neutralizing (667) IgG2a isotype monoclonal antibody (MAb). This suggested that the mobilization of adaptive immunity by administered MAbs is key for the success in the long term for the MAb-based passive immunotherapy of chronic viral infections. We have addressed here whether the anti-FrCas(E) protective endogenous immunity is the mere consequence of viral propagation blunting, which would simply give time to the immune system to react, and/or to actual immunomodulation by the MAb during the treatment. To this aim, we have compared viral replication, disease progression, and antiviral immune responses between different groups of infected mice: (i) mice treated with either the 667 MAb, its F(ab')(2) fragment, or an IgM (672) with epitopic specificity similar to that of 667 but displaying different effector functions, and (ii) mice receiving no treatment but infected with a low viral inoculum reproducing the initial viral expansion observed in their infected/667 MAb-treated counterparts. Our data show that the reduction of FrCas(E) propagation is insufficient on its own to induce protective immunity and support a direct immunomodulatory action of the 667 MAb. Interestingly, they also point to sequential actions of the administered MAb. In a first step, viral propagation is exclusively controlled by 667 neutralizing activity, and in a second one, this action is complemented by FcgammaR-binding-dependent mechanisms, which most likely combine infected cell cytolysis and the modulation of the antiviral endogenous immune response. Such complementary effects of administered MAbs must be taken into consideration for the improvement of future antiviral MAb-based immunotherapies.