Human cytomegalovirus (HCMV) possesses low pathogenic potential in an immunocompetent host. In the immunosuppressed host, however, a wide spectrum of infection outcomes, ranging from asymptomatic to life threatening, can follow either primary or nonprimary infection. The variability in the manifestations of HCMV infection in immunosuppressed individuals implies that there is a threshold of host antiviral immunity that can effectively limit disease potential. We used a nonhuman primate model of CMV infection to assess the relationship between CMV disease and the levels of developing anti-CMV immunity. Naive rhesus macaques were inoculated with rhesus cytomegalovirus (RhCMV) followed 2 or 11 weeks later by inoculation with pathogenic simian immunodeficiency virus SIVmac239. Two of four monkeys inoculated with SIV at 2 weeks after inoculation with RhCMV died within 11 weeks with simian AIDS (SAIDS), including activated RhCMV infection. Neither animal had detectable anti-SIV antibodies. The other two animals died 17 and 27 weeks after SIV inoculation with either SAIDS or early lymphoid depletion, although no histological evidence of activated RhCMV was observed. Both had weak anti-SIV antibody titers. RhCMV antibody responses for this group of monkeys were significantly below those of control animals inoculated with only RhCMV. In addition, all animals of this group had persistent RhCMV DNA in plasma and high copy numbers of RhCMV in tissues. In contrast, animals that were inoculated with SIV at 11 weeks after RhCMV infection rarely exhibited RhCMV DNA in plasma, had low copy numbers of RhCMV DNA in most tissues, and did not develop early onset of SAIDS or activated RhCMV. SIV antibody titers were mostly robust and sustained in these monkeys. SIV inoculation blunted further development of RhCMV humoral responses, unlike the normal pattern of development in control monkeys following RhCMV inoculation. Anti-RhCMV immunoglobulin G levels and avidity were slightly below control values, but levels maintained were higher than those observed following SIV infection at 2 weeks after RhCMV inoculation. These findings demonstrate that SIV produces long-lasting insults to the humoral immune system beginning very early after SIV infection. The results also indicate that anti-RhCMV immune development at 11 weeks after infection was sufficient to protect the host from acute RhCMV sequelae following SIV infection, in contrast to the lack of protection afforded by only 2 weeks of immune response to RhCMV. As previously observed, monkeys that were not able to mount a significant immune response to SIV were the most susceptible to SAIDS, including activated RhCMV infection. Rapid development of SAIDS in animals inoculated with SIV 2 weeks after RhCMV inoculation suggests that RhCMV can augment SIV pathogenesis, particularly during primary infection by both viruses.

Human cytomegalovirus (HCMV) establishes and maintains a lifelong persistence following infection in an immunocompetent host. The determinants of a stable virus-host relationship are poorly defined. A nonhuman primate model for HCMV was used to investigate virological and host parameters of infection in a healthy host. Juvenile rhesus macaques (Macaca mulatta) were inoculated with rhesus cytomegalovirus (RhCMV), either orally or intravenously (i.v.), and longitudinally necropsied. None of the animals displayed clinical signs of disease, although hematologic abnormalities were observed intermittently in i.v. inoculated animals. RhCMV DNA was detected transiently in the plasma of all animals at 1 to 2 weeks postinfection (wpi) and in multiple tissues beginning at 2 to 4 wpi. Splenic tissue was the only organ positive for RhCMV DNA in all animals. The location of splenic cells expressing RhCMV immediate-early protein 1 (IE1) in i.v. inoculated animals changed following inoculation. At 4 to 5 wpi, most IE1-positive cells were perifollicular, and at 25 wpi, the majority were located within the red pulp. All animals developed anti-RhCMV immunoglobulin M (IgM) antibodies within 1 to 2 wpi and IgG antibodies within 2 to 4 wpi against a limited number of viral proteins. Host reactivity to RhCMV proteins increased in titer (total and neutralizing) and avidity with time. These results demonstrate that while antiviral immune responses were able to protect from disease, they were insufficient to eliminate reservoirs of persistent viral gene expression.

We have generated transgenic mice that contain human-sequence Ig miniloci and, because they are also homozygous for a targeted disruption of their endogenous heavy chain genes, must rely on the transgene sequences for B cell receptor expression. Although the human transgenes contain only a fraction of the intact human heavy chain locus, these defined sequences are able to at least partially restore the humoral immune system in the mouse. B cells expressing human heavy chains develop in the bone marrow, populate peripheral lymphoid tissue and respond specifically to antigen. Furthermore, the heavy chain transgenes contain both human mu and gamma 1 coding exons as well as the respective mu and gamma 1 switch regions. The sequences included within the transgene are sufficient to direct class switch recombination. Transgene sequences are also sufficient to direct somatic mutation of the class-switched heavy chain genes. These observations define the upper limit of the cis-acting sequences necessary to direct heavy chain class switching and somatic mutation.

Human cytomegalovirus (HCMV) is a betaherpesvirus that establishes a lifelong, persistent, but asymptomatic, infection in healthy individuals. However, it can cause substantial morbidity and mortality in immunocompromised patients (1), including immunosuppressed transplant recipients and those with AIDS. HCMV is also a leading cause of birth defects after congenital infection of fetuses.Antiviral drugs approved for therapy of HCMV infection include ganciclovir (GCV), valganciclovir (vGCV), cidofovir (CDV), foscarnet (phosphonoformic acid [PFA]), and fomivirsen. GCV, vGCV, CDV, and PFA all inhibit virus replication through interaction with the viral DNA polymerase, encoded by the HCMV UL54 gene. Each of these drugs has low oral bioavailability and/or dose-related toxicities that have limited their clinical usefulness (16,31). GCV and vGCV cause substantial neutropenia and myelosuppression, and both CDV and PFA cause renal toxicity. Drug resistance is another problem in therapy with GCV, vGCV, or PFA and has been shown to occur in cell culture systems with CDV. HCMV resistance to GCV occurs predominantly from mutations in the virally encoded phosphotransferase (UL97) (9, 26) but can also arise from mutations in the DNA polymerase gene (UL54) (36). Resistance to PFA or CDV arises from mutations in UL54 (10, 11). Some of the mutations in UL54 confer cross-resistance to GCV and CDV or to GCV and PFA (10,11,34). Fomivirsen is an antisense phosphorthioate oligonucleotide that is limited to intravitreal treatment of HCMV retinitis only, whereas the other approved drugs are used systemically. There are clear clinical needs for safer anti-HCMV drugs with more favorable pharmacological properties and a need to develop new drugs that inhibit HCMV targets other than DNA polymerase.One promising class of anti-CMV compounds is the benzimidazole nucleosides (39). Unlike other nucleoside analogs, some members of this group that are active against HCMV are not phosphorylated (21) and are not substrates for DNA polymerase (22, 40). Two potent members of this class, 2,5,6-trichloro-1-(␤-D-ribofuranosyl)benzimidazole (TCRB) and its 2-bromo derivative (BDCRB), exert their activity by blocking the cleavage of concatemeric CMV DNA into genome-length pieces for packaging (40). Resistance to TCRB or BDCRB is conferred by mutations in HCMV genes UL89 and UL56 (22,40).An attractive animal model to help in the development of anti-CMV drugs is infection of rhesus macaques (Macaca mulatta) with rhesus CMV (RhCMV). This model is a strong recapitulation of the natural history of HCMV. RhCMV is endemic in populations of macaques, and immunocompetent monkeys do not display clinical signs of disease after natural infection or experimental inoculation (23, 41). RhCMV infection results in a lifelong asymptomatic persistence, characterized by periodic shedding of virus (2, 14), similar to HCMV infection of immunocompetent humans (1). However, like HCMV, RhCMV causes significant morbidity in macaques without a fully functional immune system. Fetal mac...

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