Class Switch Recombination and Somatic Hypermutation of Virus-Neutralizing Antibodies Are Not Essential for Control of Friend Retrovirus Infection

Susceptibilities to retroviral infections differ within a single host species as exemplified by the presence of slow or rapid progressors and elite controllers among individuals infected with human immunodeficiency virus (1). Friend virus (FV) infection of adult immunocompetent mice is useful to study the genetic, molecular, and cellular bases of resistance against retroviruses (2, 3). FV consists of replication-competent Friend murine leukemia virus (F-MuLV) and defective spleen focus-forming virus (SFFV). In C57BL/6 (B6) mice homozygously possessing the resistant Fv2 allele (4), not only is SFFV-induced proliferation of infected erythroid progenitor cells limited, but they are also actively eliminated by cellular immune responses (5). CD4⁺ T cells are required for SFFV elimination, while B-lymphocytes are required for F-MuLV elimination (5, 6). FV-susceptible (BALB/c × B6)F₁ (CB6F₁) mice rapidly develop splenomegaly and succumb to leukemia within 2 months postinfection (pi), but they can be protected against FV by a single immunization with an F-MuLV-encoded CD4⁺ T cell epitope, Env_462–479 (7). This vaccine-induced protection also requires B-lymphocytes, while FV-infected cells are eliminated in the absence of CD8⁺ T cells (8).

For the production of FV-neutralizing antibodies (Ab), CD11c⁺ dendritic cells and Myd88 that transduces signals from Toll-like receptors (TLR) are crucial (9). TLR7 senses retroviral entry (10) and inhibits virus replication over the first 5 days of infection through rapid production of nonneutralizing IgM (11). TLR7-deficient mice failed to generate germinal centers and IgM-negative (IgM⁻) B cells upon FV infection (12), suggesting that immunoglobulin somatic hypermutation (SHM) and class switch recombination (CSR) are associated with neutralizing Ab production and FV control. To examine directly if SHM and CSR are required for FV neutralization, we utilized mice deficient in activation-induced cytidine deaminase (AID), a B cell-specific enzyme required for SHM and CSR (13).

AID-deficient (AID^−/−) B6 and BALB/c mice were purchased from Riken BioResource Center, Tsukuba, Japan, and bred and maintained along with B cell-deficient μMT/μMT mice (5). Animal experiments were carried out in accordance with governmental and institutional regulations. To examine if SHM and CSR are required for Fv2-associated resistance, we infected wild-type (WT), μMT/μMT, and AID^−/− B6 mice with 5,000 spleen focus-forming units (SFFU) of FV that was free of lactate dehydrogenase-elevating virus (5). All μMT/μMT mice died within 20 weeks pi (Fig. 1A) without developing polycythemia (Fig. 1C), while AID^−/− mice remained resistant. F-MuLV gp70 expression on expanding TER-119⁻ cells consistent with the development of F-MuLV-induced myeloid leukemia (5) was evident in μMT/μMT but not in AID^−/− mice (Fig. 1B). Thus, AID-mediated SHM and CSR are not required for F-MuLV elimination. Plasma viremia and virus-neutralizing Ab were detected as described previously (14). While μMT/μMT mice remained viremic even at 16 weeks pi, AID^−/− mice cleared viremia, albeit less quickly than the WT mice did (Fig. 1D). As virus particles in the plasma might not be detected by the above-described focus-formation assays in the presence of antiviral Ab, viral RNA was extracted from 20 μl of each plasma sample using an RNeasy Plus kit (Qiagen, Valencia, CA) and eluted into 40 μl of RNase-free water, and 11 μl of each eluate was used for cDNA synthesis using the oligo(dT)₂₀ primer and a SuperScript III first-strand synthesis system (Life Technologies, Carlsbad, CA). Real-time PCR assays for the quantification of the F-MuLV genome were performed as described previously (5). At 4 weeks pi, the level of the F-MuLV genome was below the limit of detection in 4 of the 5 infected WT mice, while low levels of the F-MuLV genome were detectable in the plasma samples from infected AID^−/− mice (Fig. 1D, right panel). However, the average plasma number of F-MuLV genome was significantly lower in infected AID^−/− than in the μMT/μMT mice and was not different from the number seen in the WT mice. Thus, AID^−/− B6 mice can control viremia. AID^−/− mice possessed high titers of virus-neutralizing Ab at 3 weeks pi (Fig. 1E), clearly indicating that IgM produced in the absence of AID can neutralize F-MuLV.

We next examined if SHM and CSR are required for vaccine-induced protection of FV-susceptible mice. As previously described (7, 8), WT CB6F₁ mice were 100% protected by a single peptide immunization, while μMT/μMT mice died regardless of the presence or absence of vaccination. About half of the vaccinated AID^−/− mice were free from polycythemia and survived past 12 weeks pi (Fig. 2A and B), indicating that SHM and CSR may contribute to but are not absolutely required for peptide-induced protection of CB6F₁ mice against FV. To confirm that AID^−/− mice can control FV after peptide immunization, we reduced the challenge dose to 10 SFFU, which still killed most of the unimmunized CB6F₁ mice by 12 weeks pi (Fig. 2C). While no effect of peptide immunization was observed in μMT/μMT mice, 90% of AID^−/− mice were protected against the low-dose challenge. Levels of viremia determined by both fluorescence focus assays and the enumeration of F-MuLV genomic RNAs and numbers of FV infectious centers were all significantly lower in peptide-immunized than in unimmunized animals even in the absence of AID (Fig. 3A and B), and virus-neutralizing IgM became detectable in immunized AID^−/− mice as quickly as in the WT mice (Fig. 3C).

To examine directly if IgM produced in the absence of AID can contribute to FV control, passive transfer was attempted. AID^−/− CB6F₁ mice were subjected to peptide immunization and challenged with FV, and sera were collected at 3 weeks pi and subjected to heat inactivation (14). To reproduce Ab production in infected mice, we examined the kinetics of anti-FV Ab production. F-MuLV particles were purified (15) and adsorbed onto a nitrocellulose membrane (Multiscreen-HA; Millipore, Bedford, MA). A serially diluted spleen cell suspension was added to the membrane-bottomed wells and incubated for 18 h, followed by Ab detection with horseradish peroxidase-conjugated anti-mouse IgM (SouthernBiotech, Birmingham, AL) and 3-amino-9-ethylcarbazole. In peptide-immunized AID^−/− mice, cells producing anti-F-MuLV IgM were detected at 3 days pi, and the cell numbers became significantly higher than those in unimmunized mice at 7 days pi (Fig. 4A). F-MuLV-neutralizing serum Ab started to be detected in peptide-immunized AID^−/− mice at 6 days pi with an average titer of 2^2.5 (n = 5). Therefore, we started injecting B cell-deficient CB6F₁ mice with the pooled sera on day 5 pi (Fig. 4B). Prior to serum transfer, recipients were either immunized with the peptide or given adjuvant alone and challenged with 150 SFFU FV 3 weeks later. Peptide-immunized μMT/μMT recipient mice given immune sera from the WT mice possessed undetectable or very low levels of FV infectious centers and normal spleen sizes at 4 weeks pi, indicating the efficacy of the adoptive immunization described above (data not shown). Immunized mice given naive sera all died by 7 weeks pi, while a significantly higher proportion of those given the immune sera from AID^−/− mice survived past 12 weeks pi (Fig. 4B), reproducing the partial protection of immunized AID^−/− mice (Fig. 2B). A previous report demonstrated the requirement of host T cells for passive immunotherapy with FV-neutralizing monoclonal IgG (16). Similarly, no protection was observed when the recipients were not previously immunized. Serum transfer on day 14 pi alone was ineffective.

As a recent report (17) has indicated that an apolipoprotein B mRNA editing enzyme, catalytic polypeptide 3 ([APOBEC3]), expressed in B6 mice can complement AID in generating SHM and as we performed the present study in the presence of B6-derived APOBEC3, the IgM Ab we detected in AID^−/− mice might not necessarily have been unmutated, although whether APOBEC3 alone induces significant SHM in the absence of AID has not been examined. Nevertheless, it is now clear that, although TLR7-Myd88 signaling is required for anti-FV Ab production and germinal center responses (9–12), AID-mediated SHM and CSR are not absolutely required, and IgM produced in the absence of AID can still contribute to spontaneous and vaccine-induced resistance to FV infection.

Animal experiments and flow cytometry were supported by members of the Central Research Facilities, Kinki University Faculty of Medicine. AID-deficient B6 (BRC 00897) and BALB/c (BRC 00901) mice were kindly provided by Tasuku Honjo through the Riken BioResource Center with the support of the National BioResource Project of the Ministry of Education, Culture, Sports, Science and Technology of Japan (MEXT). We thank J. B. Dowell for critical reading and correction of the manuscript.

This work was supported in part by Grants-in-Aid for Scientific Research (KAKENHI and the Anti-aging Center Project) from MEXT and those from the Ministry of Health, Labor and Welfare of Japan for research on HIV/AIDS.