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 FcγR-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.Although monoclonal antibodies (MAbs) principally have been considered for anticancer applications heretofore (62, 64), they now are increasingly being considered to treat severe acute and chronic viral infections (43, 63, 83). The best-studied antiviral MAbs are (i) pavalizumab, a humanized anti-respiratory syncytial virus (RSV) MAb approved by the FDA in 1998 for treating severe lower-respiratory-tract diseases in infants (45); (ii) several anti-human immunodeficiency virus (HIV) MAbs, which have been used in macaque preclinical infection models and in several human trials (4, 5, 19, 27-30, 32, 42, 50, 55, 57, 76-79); and (iii) a few anti-hepatitis C virus (HCV) MAbs, some of which currently are being tested in humans (9, 22, 40). However, other MAbs, some of them of human origin, also have been generated against other human viruses in recent years. Among them are antibodies against Ebola virus (75), West Nile virus (WNV) (48, 53, 54), cytomegalovirus (CMV) (11), avian and human influenza viruses (59, 60, 73, 74), severe acute respiratory syndrome coronavirus (SARS CoV) (81), hepatitis B virus (HBV) (31, 35), Hanta virus (80, 82), and Nipah virus (80, 82). These antiviral MAbs all have been selected on the basis of their neutralizing activity and the possibility that they interfere with the antiviral immune response of treated hosts, because their effector functions have been considered surprisingly little so far. Addressing this question in clinical settings currently is not possible for a variety of reasons that include ethical, technical, and cost concerns. Therefore, we have turned to the neonatal infection of mice by the lethal FrCas^E retrovirus as a model system. This model allowed us to show that a very short immunotherapy by a neutralizing MAb of the IgG2a isotype (667 MAb) can permit, in addition to an immediate direct effect on the viral load, the mounting of a long-lasting endogenous antiviral immunity, which is essential for viral control and healthy survival (23-25). Because of the broad therapeutic perspectives opened by this observation, it now is essential to elucidate the molecular and cellular mechanisms underlying this effect.FrCas^E is a simple chimeric mouse retrovirus in which the env gene of the leukemogenic Friend murine leukemia virus (F-MuLV) was replaced by that of the neurodegeneration-inducing CasBr retrovirus (58). When 5 × 10⁴ infectious particles are inoculated into newborn mice under the age of 5 to 6 days, FrCas^E can enter the central nervous system (CNS) and induces a neurodegeneration fatal within 1 to 2 months with 100% incidence (15, 23, 41, 58). However, upon infection at a later time, FrCas^E can no longer enter the CNS. Instead, it replicates only in the periphery and gives rise to a fatal erythroleukemia preceded by spleen enlargement and a dramatic drop of the hematocrit. Erythroleukemia incidence and incubation period, however, are variable, depending on the inoculum and the date of infection (46).667 is an IgG2a/κ (44) directed to the main viral receptor-binding site of CasBr Env (16). It displays both in vitro (44) and in vivo (56) neutralizing activities. When rapidly (<2 days) administered for a few days to neonatally FrCas^E-infected pups, viral propagation is rapidly blunted, which prevents virus entry in the brain and subsequent neurodegeneration (23). Moreover, all 667-treated mice develop a strong, long-lasting antiviral immune response, which is necessary for them to survive healthy and with no sign of neurodegeneration or of erythroleukemia (23-25) and to resist viral challenges carried out as long as 14 months after first infection (23). Protective antiviral immunity is of a typical TH1 type with humoral and cytotoxic T-cell (CTL) contributions. The anti-FrCas^E humoral contribution is high, sustained, and principally of the IgG2a type with both in vitro neutralization- and complement-dependent cytolysis activities (23). Interestingly, it shows typical secondary response characteristics in viral challenge experiments (23), and anti-FrCas^E antibodies are transmitted transplacentally and through breastfeeding by mothers to children, where they manifest the same properties as those of 667 in the perinatal infection setting, i.e., they prevent mice from developing neurodegeneration and permit the induction of an endogenous protective antiviral immune response (25). Finally, the CTL response directed to infected cells was shown to be necessary for the protection of FrCas^E-infected, 667-treated mice, as the depletion of CD8⁺ T cells leads to death by retrovirally induced erythroleukemia (24).At this stage, an important issue is the clarification of whether the anti-FrCas^E protective immunity seen in 667 MAb-treated mice is due to actual immunomodulation by the MAb owing to its effector function(s) and/or is the consequence of viral propagation blunting, which would prevent the immune system from being overwhelmed by an excess of antigen and, hence, would give it time to react optimally. To address these two nonexclusive possibilities, we compared here viral propagations, health statuses, and endogenous immune responses in four groups of mice. The first three groups were mice neonatally infected under standard conditions (5 × 10⁴ infectious particles) and treated with either the natural 667 MAb, the antibody effector function-lacking F(ab′)₂ fragment of 667, or a neutralizing IgM (672) with effector functions inherently different from those of 667. The last group consisted of mice neonatally infected with a low FrCas^E inoculum but not subjected to immunotherapy, which is a condition permitting early viral propagation kinetics similar to those of animals infected and 667 MAb treated under standard conditions. Taken together, our data indicate that the drastic reduction of viral propagation shortly after infection is not sufficient for the induction of protective adaptive immunity and, thereby, point to an immunomodulatory action of 667. Interestingly, they also point to two sequential actions of the administered MAb. In the immediate postinfection period, viral spread is controlled exclusively by 667 neutralizing activity, and later it involves the cytolysis of infected cells owing to FcγR-binding-dependent mechanisms. Finally, our work shows that not all antibody isotypes are equally efficient at protecting infected mice and favoring the mounting of protective immunity, as 672 IgM immunotherapy-treated animals died of erythroleukemia.