Analysis of Host Range Restriction Determinants in the Rabbit Model: Comparison of Homologous and Heterologous Rotavirus Infections

The main limitation of both the rabbit and mouse models of rotavirus infection is that human rotavirus (HRV) strains do not replicate efficiently in either animal. The identification of individual genes necessary for conferring replication competence in a heterologous host is important to an understanding of the host range restriction of rotavirus infections. We recently reported the identification of the P type of the spike protein VP4 of four lapine rotavirus strains as being P[14]. To determine whether VP4 is involved in host range restriction in rabbits, we evaluated infection in rotavirus antibody-free rabbits inoculated orally with two P[14] HRVs, PA169 (G6) and HAL1166 (G8), and with several other HRV strains and animal rotavirus strains of different P and G types. We also evaluated whether the parental rhesus rotavirus (RRV) (P5B[3], G3) and the derived RRV-HRV reassortant candidate vaccine strains RRV × D (G1), RRV × DS-1 (G2), and RRV × ST3 (G4) would productively infect rabbits. Based on virus shedding, limited replication was observed with the P[14] HRV strains and with the SA11 Cl3 (P[2], G3) and SA11 4F (P6[1], G3) animal rotavirus strains, compared to the homologous ALA strain (P[14], G3). However, even limited infection provided complete protection from rotavirus infection when rabbits were challenged orally 28 days postinoculation (DPI) with 10³ 50% infective doses of ALA rabbit rotavirus. Other HRVs did not productively infect rabbits and provided no significant protection from challenge, in spite of occasional seroconversion. Simian RRV replicated as efficiently as lapine ALA rotavirus in rabbits and provided complete protection from ALA challenge. Live attenuated RRV reassortant vaccine strains resulted in no, limited, or productive infection of rabbits, but all rabbits were completely protected from heterotypic ALA challenge. The altered replication efficiency of the reassortants in rabbits suggests a role for VP7 in host range restriction. Also, our results suggest that VP4 may be involved in, but is not exclusively responsible for, host range restriction in the rabbit model. The replication efficiency of rotavirus in rabbits also is not controlled by the product of gene 5 (NSP1) alone, since a reassortant rotavirus with ALA gene 5 and all other genes from SA11 was more severely replication restricted than either parental rotavirus strain.Rotaviruses are the leading cause of acute viral gastroenteritis in humans and animals throughout the world. Rotaviruses belong to the Reoviridae family and are characterized by a genome consisting of 11 segments of double-stranded RNA (dsRNA), enclosed in a triple-layered protein capsid (28). Serotype designations are based on independent neutralization determinants on the two outer capsid proteins VP4 (P serotypes, for protease-sensitive protein) and VP7 (G serotypes, for glycoprotein) (28). Serotype specificity determined by cross-neutralization assays using hyperimmune sera against the whole virus is mainly defined by VP7, and 14 G serotypes have been identified (28). Recently, antisera or monoclonal antibodies raised to VP4 and sequence analysis of VP4 identified 12 P serotypes and 20 P genotypes, respectively (28, 39). Rotavirus VP4 protein is responsible for a number of important biological functions, such as the enhancement of infectivity by proteolytic cleavage of VP4 into VP8* and VP5*, hemagglutination, restricted growth in cell culture, virulence, initial virus attachment to cells, and protease sensitivity associated with plaque formation (1, 4, 25, 34, 40, 51).The use of animal models, including the rabbit and mouse models, has been essential to the understanding of rotavirus infection, pathology, disease, immunity, and testing of prospective vaccines in children (21). The limitations of the rabbit and adult mouse models of rotavirus infection for vaccine testing are as follows: (i) human rotavirus (HRV) strains do not efficiently replicate in either animal, (ii) clinical disease is not observed, and (iii) only homologous virus strains (isolated from the same species) replicate efficiently and spread horizontally to uninoculated control animals, whereas heterologous virus strains (isolated from a different species) do not (6, 15, 16, 29, 31, 35, 37, 44, 50, 55). We and others developed a rabbit model of rotavirus infection that is useful for defining basic parameters of active immunity, immunogenicity, and protective efficacy of vaccines (12, 15–21, 36, 61). Rabbits are productively infected with homologous lapine rotavirus strains up to at least the age of 5 years, which allows examination of active and long-term immunity for vaccine studies (13, 15–17, 36, 61). Group A lapine rotavirus strains have been isolated in Canada, Japan, Italy, and the United States, and those that have been characterized are serotype G3 (8, 11, 15, 53, 56, 61). Recently, the P type of four different strains was identified as genotype P[14] (11). Previously, limited infection of rabbits with a heterologous strain had been obtained only with SA11 Cl3 (P[2], G3) (15).Attempts to identify host range and virulence determinants for rotavirus have implicated different constellations of genes, including genes 2, 3, 4, 5, 8, 9, 10, and 11 (5, 23, 30, 33, 37, 38, 41, 43, 44, 60, 62, 65). Although host range restriction and virulence may be multigenic, two genes, 4 and 5, are of interest because they cluster according to species of origin, suggesting a role in host range restriction. The finding that genome segment 5 (NSP1) sequences cluster according to species of origin (24, 39, 65) and that, in the mouse model, gene 5 segregates with transmission of virus among littermates (5), led to the hypothesis that NSP1 is involved in host range restriction. VP4 sequence analyses of rotavirus strains isolated from different species revealed that specific VP4 types also generally correlate with the species of origin of each rotavirus strain (43, 60). Therefore, once we identified the P type of four lapine rotaviruses as P[14], we tested two P[14] HRV strains, PA169 (G6) and HAL1166 (G8) (32) to determine if VP4 is involved in host range restriction. We also tested several other HRV strains, live attenuated reassortant candidate vaccine strains [rhesus rotavirus (RRV) × D (G1), RRV × DS-1 (G2), and RRV × ST3 (G4)], and animal rotavirus strains of different P and G types to determine if they could productively infect rabbits. In addition, to evaluate whether the single rotavirus gene 5 is responsible for replication efficiency in rabbits, rabbits were inoculated with a reassortant rotavirus with the lapine ALA gene 5 and all the other genes from the simian rotavirus SA11 Cl3 strain.     

Enhanced Virus Clearance by Early Inducible Lymphocytic Choriomeningitis Virus-Neutralizing Antibodies in Immunoglobulin-Transgenic Mice

Following infection of mice with lymphocytic choriomeningitis virus (LCMV), virus-neutralizing antibodies appear late, after 30 to 60 days. Such neutralizing antibodies play an important role in protection against reinfection. To analyze whether a neutralizing antibody response which developed earlier could contribute to LCMV clearance during the acute phase of infection, we generated transgenic mice expressing LCMV-neutralizing antibodies. Transgenic mice expressing the immunoglobulin μ heavy chain of the LCMV-neutralizing monoclonal antibody KL25 (H25 transgenic mice) mounted LCMV-neutralizing immunoglobulin M (IgM) serum titers within 8 days after infection. This early inducible LCMV-neutralizing antibody response significantly improved the host’s capacity to clear the infection and did not cause an enhancement of disease after intracerebral (i.c.) LCMV infection. In contrast, mice which had been passively administered LCMV-neutralizing antibodies and transgenic mice exhibiting spontaneous LCMV-neutralizing IgM serum titers (HL25 transgenic mice expressing the immunoglobulin μ heavy and the κ light chain) showed an enhancement of disease after i.c. LCMV infection. Thus, early-inducible LCMV-neutralizing antibodies can contribute to viral clearance in the acute phase of the infection and do not cause antibody-dependent enhancement of disease.Against many cytopathic viruses such as poliovirus, influenza virus, rabies virus, and vesicular stomatitis virus, protective virus-neutralizing antibodies are generated early, within 1 week after infection (3, 31, 36, 44, 49). In contrast, several noncytopathic viruses (e.g., human immunodeficiency virus and hepatitis viruses B and C in humans or lymphocytic choriomeningitis virus [LCMV] in mice) elicit poor and delayed virus-neutralizing antibody responses (1, 7, 20, 24, 27, 35, 45, 48).In the mouse, the natural host of LCMV, the acute LCMV infection is predominantly controlled by cytotoxic T lymphocytes (CTLs) in an obligatory perforin-dependent manner (13, 18, 28, 50). In addition to the CTL response, LCMV-specific antibodies are generated. Early after infection (by day 8), a strong antibody response specific for the internal viral nucleoprotein (NP) is mounted (7, 19, 23, 28). These early LCMV NP-specific antibodies exhibit no virus-neutralizing capacity (7, 10). Results from studies of B-cell-depleted mice and B-cell-deficient mice implied that the early LCMV NP-specific antibodies are not involved in the clearance of LCMV (8, 11, 12, 40). Late after infection (between days 30 and day 60), LCMV-neutralizing antibodies develop (7, 19, 22, 28, 33); these antibodies are directed against the surface glycoprotein (GP) of LCMV (9, 10). LCMV-neutralizing antibodies have an important function in protection against reinfection (4, 6, 38, 41, 47).In some viral infections, subprotective virus-neutralizing antibody titers can enhance disease rather than promote host recovery (i.e., exhibit antibody-dependent enhancement of disease [ADE] [14, 15, 21, 46]). For example, neutralizing antibodies are involved in the resolution of a primary dengue virus infection and in the protection against reinfection. However, if subprotective neutralizing antibody titers are present at the time of reinfection, a severe form of the disease (dengue hemorrhagic fever/dengue shock syndrome [15, 21]), which might be caused by Fc receptor-mediated uptake of virus-antibody complexes leading to an enhanced infection of monocytes (15, 16, 25, 39), can develop. Similarly, an enhancement of disease after intracerebral (i.c.) LCMV infection was observed in mice which had been treated with virus-neutralizing antibodies before the virus challenge (6). ADE in LCMV-infected mice was either due to an enhanced infection of monocytes by Fc receptor-mediated uptake of antibody-virus complexes or due to CTL-mediated immunopathology caused by an imbalanced virus spread and CTL response.To analyze whether LCMV-neutralizing antibodies generated early after infection improve the host’s capacity to clear the virus or enhance immunopathological disease, immunoglobulin (Ig)-transgenic mice expressing LCMV-neutralizing IgM antibodies were generated. After LCMV infection of transgenic mice expressing the Ig heavy chain (H25 transgenic mice), LCMV-neutralizing serum antibodies were mounted within 8 days, which significantly improved the host’s capacity to eliminate LCMV. H25 transgenic mice did not show any signs of ADE after i.c. LCMV infection.Transgenic mice expressing the Ig heavy and light chains (HL25 transgenic mice) exhibited spontaneous LCMV-neutralizing serum antibodies and confirmed the protective role of preexisting LCMV-neutralizing antibodies, even though the neutralizing serum antibodies were of the IgM isotype. Similar to mice which had been treated with LCMV-neutralizing antibodies, HL25 transgenic mice developed an enhanced disease after i.c. LCMV infection, which indicated that ADE was due to an imbalance between virus spread and CTL response. Thus, the early-inducible LCMV-neutralizing antibody response significantly enhanced clearance of the acute infection without any risk of causing ADE.     

The Cell Tropism of Human Immunodeficiency Virus Type 1 Determines the Kinetics of Plasma Viremia in SCID Mice Reconstituted with Human Peripheral Blood Leukocytes

Most individuals infected with human immunodeficiency virus type 1 (HIV-1) initially harbor macrophage-tropic, non-syncytium-inducing (M-tropic, NSI) viruses that may evolve into T-cell-tropic, syncytium-inducing viruses (T-tropic, SI) after several years. The reasons for the more efficient transmission of M-tropic, NSI viruses and the slow evolution of T-tropic, SI viruses remain unclear, although they may be linked to expression of appropriate chemokine coreceptors for virus entry. We have examined plasma viral RNA levels and the extent of CD4⁺ T-cell depletion in SCID mice reconstituted with human peripheral blood leukocytes following infection with M-tropic, dual-tropic, or T-tropic HIV-1 isolates. The cell tropism was found to determine the course of viremia, with M-tropic viruses producing sustained high viral RNA levels and sparing some CD4⁺ T cells, dual-tropic viruses producing a transient and lower viral RNA spike and extremely rapid depletion of CD4⁺ T cells, and T-tropic viruses causing similarly lower viral RNA levels and rapid-intermediate rates of CD4⁺ T-cell depletion. A single amino acid change in the V3 region of gp120 was sufficient to cause one isolate to switch from M-tropic to dual-tropic and acquire the ability to rapidly deplete all CD4⁺ T cells.The envelope gene of human immunodeficiency virus type 1 (HIV-1) determines the cell tropism of the virus (11, 32, 47, 62), the use of chemokine receptors as cofactors for viral entry (4, 17), and the ability of the virus to induce syncytia in infected cells (55, 60). Cell tropism is closely linked to but probably not exclusively determined by the ability of different HIV-1 envelopes to bind CD4 and the CC or the CXC chemokine receptors and initiate viral fusion with the target cell. Macrophage-tropic (M-tropic) viruses infect primary cultures of macrophages and CD4⁺ T cells and use CCR5 as the preferred coreceptor (2, 5, 15, 23, 26, 31). T-cell-tropic (T-tropic) viruses can infect primary cultures of CD4⁺ T cells and established T-cell lines, but not primary macrophages. T-tropic viruses use CXCR4 as a coreceptor for viral entry (27). Dual-tropic viruses have both of these properties and can use either CCR5 or CXCR4 (and infrequently other chemokine receptors [25]) for viral entry (24, 37, 57). M-tropic viruses are most frequently transmitted during primary infection of humans and persist throughout the duration of the infection (63). Many, but not all, infected individuals show an evolution of virus cell tropism from M-tropic to dual-tropic and finally to T-tropic with increasing time after infection (21, 38, 57). Increases in replicative capacity of viruses from patients with long-term infection have also been noted (22), and the switch to the syncytium-inducing (SI) phenotype in T-tropic or dual-tropic isolates is associated with more rapid disease progression (10, 20, 60). Primary infection with dual-tropic or T-tropic HIV, although infrequent, often leads to rapid disease progression (16, 51). The viral and host factors that determine the higher transmission rate of M-tropic HIV-1 and the slow evolution of dual- or T-tropic variants remain to be elucidated (4).These observations suggest that infection with T-tropic, SI virus isolates in animal model systems with SCID mice grafted with human lymphoid cells or tissue should lead to a rapid course of disease (1, 8, 44–46). While some studies in SCID mice grafted with fetal thymus and liver are in agreement with this concept (33, 34), our previous studies with the human peripheral blood leukocyte-SCID (hu-PBL-SCID) mouse model have shown that infection with M-tropic isolates (e.g., SF162) causes more rapid CD4⁺ T-cell depletion than infection with T-tropic, SI isolates (e.g., SF33), despite similar proviral copy numbers, and that this property mapped to envelope (28, 41, 43). However, the dual-tropic 89.6 isolate (19) caused extremely rapid CD4⁺ T-cell depletion in infected hu-PBL-SCID mice that was associated with an early and transient increase in HIV-1 plasma viral RNA (29). The relationship between cell tropism of the virus isolate and the pattern of disease in hu-PBL-SCID mice is thus uncertain. We have extended these studies by determining the kinetics of HIV-1 RNA levels in serial plasma samples of hu-PBL-SCID mice infected with primary patient isolates or laboratory stocks that differ in cell tropism and SI properties. The results showed significant differences in the kinetics of HIV-1 replication and CD4⁺ T-cell depletion that are determined by the cell tropism of the virus isolate.     

A Peptide Mimic of a Protective Epitope of Respiratory Syncytial Virus Selected from a Combinatorial Library Induces Virus-Neutralizing Antibodies and Reduces Viral Load In Vivo

Respiratory syncytial virus (RSV) is the most important cause of bronchiolitis and pneumonia in infants and young children worldwide. As yet, there is no effective vaccine against RSV infection, and previous attempts to develop a formalin-inactivated vaccine resulted in exacerbated disease in recipients subsequently exposed to the virus. In the work described here, a combinatorial solid-phase peptide library was screened with a protective monoclonal antibody (MAb 19) to identify peptide mimics (mimotopes) of a conserved and conformationally-determined epitope of RSV fusion (F) protein. Two sequences identified (S1 [HWYISKPQ] and S2 [HWYDAEVL]) reacted specifically with MAb 19 when they were presented as solid-phase peptides. Furthermore, after amino acid substitution analyses, three sequences derived from S1 (S1S [HWSISKPQ], S1K [KWYISKPQ], and S1P [HPYISKPQ]), presented as multiple antigen peptides (MAPs), also showed strong reactivity with MAb 19. The affinity constants of the binding of MAb 19, determined by surface plasmon resonance analyses, were 1.19 × 10⁹ and 4.93 × 10⁹ M⁻¹ for S1 and S1S, respectively. Immunization of BALB/c mice with these mimotopes, presented as MAPs, resulted in the induction of anti-peptide antibodies that inhibited the binding of MAb 19 to RSV and neutralized viral infection in vitro, with titers equivalent to those in sera from RSV-infected animals. Following RSV challenge of S1S mimotope-immunized mice, a 98.7% reduction in the titer of virus in the lungs was observed. Furthermore, there was a greatly reduced cell infiltration in the lungs of immunized mice compared to that in controls. These results indicate the potential of peptide mimotopes to protect against RSV infection without exacerbating pulmonary pathology.Respiratory syncytial virus (RSV) is the major cause of serious lower respiratory tract illness in infants and immunosuppressed individuals worldwide and is estimated to be responsible for 65 million infections and 1 million deaths annually (12, 19). Although the severity of disease declines with repeated infection, previous infection with RSV does not prevent illness in subsequent infections, and it is apparent that immunity is incomplete. Furthermore, attempts to develop a vaccine against RSV have encountered a series of problems. In the late 1960s, a formalin-inactivated vaccine not only failed to protect infants against RSV but also induced exacerbated disease during a subsequent epidemic (5, 19). Retrospective analysis of the sera demonstrated that the inactivated vaccine induced high anti-F (fusion) protein antibody titers, but with poor neutralizing activity, suggesting that the inactivation treatment had denatured or modified epitopes which were the target for neutralizing antibodies (19, 25). Live attenuated vaccines were also investigated as candidates; however, these were either poorly immunogenic (overattenuated) or genetically unstable (5, 45). Although new attenuated vaccines have given encouraging results in animal models (10, 11), there is an urgent need for alternative approaches to the development of an effective vaccine.Studies with experimental animals have provided evidence that RSV-specific neutralizing antibodies can prevent infection in the lungs when administered prophylactically (29). Intravenous administration of pooled human immunoglobulin G (IgG) containing a high titer of neutralizing antibodies prevented serious RSV lower respiratory tract illness in high-risk children (18). Both the F and G (attachment) glycoproteins of RSV play a major role in eliciting this humoral immunity. The conserved F glycoprotein induces protective immune responses (8, 36), and passive immunization with neutralizing anti-F monoclonal antibodies (MAbs) or recombinant Fab protects small animals from RSV infection (12, 38, 39). Based on the results of these studies, a neutralizing and protective MAb (MAb 19) has been reshaped and humanized (as MAb RSHZ19) and has been demonstrated to have protective properties superior to those of anti-RSV polyclonal antibodies in a rodent model (46). Its safety and efficacy have recently been assessed in human volunteers (13).The immunochemical characterization of the epitopes recognized by these protective antibodies is critical for the development of a vaccine against RSV, but the conformational constraints associated with the protective epitopes in RSV F protein make it unlikely that they will be identified from analysis of the primary amino acid sequence. Indeed, earlier attempts in our laboratory and elsewhere with synthetic peptides representing continuous sequences of the F protein (amino acids [aa] 205 to 225, 221 to 237, 261 to 273, 215 to 275, 417 to 438, and 481 to 491) failed to generate protective humoral responses (4, 23, 32, 42). The use of peptides (3), antigen fragments (24), and antibody escape mutants (39) has confirmed the involvement of discontinuous residues within the F protein in epitopes recognized by protective MAbs (39). Thus, the use of alternative approaches for the identification of these epitopes is essential.Solid-phase and bacteriophage combinatorial peptide libraries (22, 31) have been used for identification of peptide mimics (mimotopes) of ligands, and the identification of peptide sequences that mimic the conformational structure of protective epitopes would have great potential for the development of a synthetic peptide vaccine. Recently, using a solid-phase combinatorial peptide library, we identified 8-mer peptides that mimic an epitope recognized by a monoclonal anti-measles virus F protein antibody. The mimotopes identified by this approach did not bear any primary sequence relationship to sequences in the viral protein but mimicked its conformation. When used as an immunogen, one of the mimotopes induced virus-neutralizing and protective antibody responses (35). Phage-displayed peptide libraries have been used to delineate sequences which mimic a discontinuous epitope of hepatitis B surface antigen (7), the secondary structure of a neutralizing epitope of human immunodeficiency virus type 1 (15), and peptides that reacted with MAbs specific for polysaccharide antigens (43).In this paper, we describe the identification of mimotopes of a protective epitope of RSV F protein, as defined by the protective MAb 19, following the screening of a solid-phase combinatorial peptide library. One of these mimotopes induced a virus-neutralizing antibody response equivalent to that in RSV-immunized animals, which significantly reduced the viral load in RSV-challenged mice. These findings indicate the potential of synthetic peptide mimotopes for the development of a novel vaccine against RSV.