Lessons from studies with murine cytomegalovirus that could lead to a safe live attenuated vaccine for human cytomegalovirus.

Studies with a murine cytomegalovirus mutant suggested two possible approaches to producing a live attenuated human cytomegalovirus vaccine. One approach would be to use a combination of five to six mutants where an attenuating mutation in the gene of one mutant is compensated by the wild-type version in a second mutant, which in turn has a mutation in a different gene compensated by the wild-type version in a third mutant, etc. Important genes in this approach could include those involved in DNA replication.

Further studies on the role of the residue 890 cysteine to tyrosine mutation in the M70 primase ORF of the temperature-sensitive mutant (tsm5) of murine cytomegalovirus.

A mutation (C890Y) introduced into the M70 primase gene of murine cytomegalovirus (MCMV) resulted in reduced viral replication in murine embryo fibroblasts at 40°C and the mutant was severely attenuated in vivo. The attenuated replication of the M70 mutant was also observed in Raw 264.7 macrophages at 37°C, demonstrating that the mutation produced a defective rather than an unstable protein possibly reducing the amount of functional protein under different environmental conditions.

An attenuated temperature-sensitive strain of cytomegalovirus (tsm5) establishes immunity without development of CD8(+) T cell memory inflation.

Cytomegalovirus (CMV) is a widely prevalent herpesvirus that is well tolerated by an immune competent host yet establishes a state of chronic infection. The virus is thought to undergo frequent subclinical episodes of reactivation which leads to an unusually large accumulation of CMV-specific CD8(+) T lymphocytes in the peripheral blood, a phenomenon termed "memory inflation." The high magnitude of the CMV T cell response has been implicated in impaired immunity to heterologous pathogens such as EBV, influenza and West Nile virus.

Antiviral therapy can reverse the development of immune senescence in elderly mice with latent cytomegalovirus infection.

Cytomegalovirus (CMV) infection leads to the development of adaptive and humoral immune responses that are among the largest for any pathogen, and intriguingly, the magnitude of the immune response increases with age, a phenomenon termed "memory inflation." Elevated CMV-specific immunity has been correlated with an increased mortality rate in elderly individuals and with impaired vaccination responses. The latent phase of CMV infection is characterized by intermittent episodes of subclinical viral reactivation and the production of immunogenic transcripts that may maintain memory inflation of virus-specific cytotoxic lymphocytes.

Role of mutations identified in ORFs M27, M36, m139, m141, and m143 in the temperature-sensitive phenotype of murine cytomegalovirus mutant tsm5.

A mutant of murine cytomegalovirus (MCMV), tsm5, which is temperature-sensitive for replication in murine embryo fibroblasts at 40°C, failed to replicate to detectable levels in mice. A total of 18 non-synonymous mutations have been identified in tsm5. In a previous study, a mutation (C890Y) identified in the M70 primase gene, when introduced into the wt M70 primase, resulted in a mutant with reduced viral replication at 40°C in vitro and which was severely attenuated in vivo.

NK cells protect secondary lymphoid tissue from cytomegalovirus via a CD30-dependent mechanism.

The pathogenic outcomes of viral infection are often reminiscent of a dysfunctional immune system. Thus, cytomegalovirus (CMV) causes disruption of the lymphoid architecture and the functionality of lymphocytes, both of which are features of CD30 deficiency. It was therefore plausible that CD30 might interfere with CMV infection.

Role of mutations identified in ORFs M56 (terminase), M70 (primase) and M98 (endonuclease) in the temperature-sensitive phenotype of murine cytomegalovirus mutant tsm5.

Twenty-six non-synonymous and synonymous mutations have been identified in the temperature-sensitive (ts) mutant (tsm5) of the K181 (Birmingham) variant of murine cytomegalovirus that is deficient in DNA synthesis, processing and packaging at the non-permissive temperature and produces undetectable levels of infectious virus in mice. Non-synonymous mutations identified in the M70 (primase), M56 (terminase) and M98 (nuclease) ORFs were introduced individually and in combination into the K181 (Perth) variant using BAC technology to examine their role in the ts phenotype. The M56 (G439R) and M98 (P324S) mutations had no evident role in the ts phenotype.

Synergistic OX40 and CD30 signals sustain CD8+ T cells during antigenic challenge.

Prior to acquiring a memory phenotype, antigen-activated CD8(+) T cells need to expand and then undergo a contraction phase. Utilizing two different antigenic stimuli, we provide evidence that the tumor necrosis factor receptors OX40 and CD30 integrate synergistic signals during the expansion phase to help maintain CD8(+) effectors. Thus, double deficiency in OX40 and CD30 leads to CD8(+) cell loss during expansion after immunization either with OVA or with murine CMV.

Identification of mutations in a temperature-sensitive mutant (tsm5) of murine cytomegalovirus using complementary genome sequencing.

Identification of mutations in mutants derived chemically is a difficult and relatively random process. NimbleGen Comparative Genome Sequencing (CGS) was assessed as an inexpensive, rapid method of identifying mutations in the temperature-sensitive mutant tsm5 of the K181 (Birmingham) variant of murine cytomegalovirus (MCMV). This genome resequencing approach requires an established genome sequence as a reference.

Preliminary characterization of murine cytomegaloviruses with insertional and deletional mutations in the M34 open reading frame.

A viable virus could not be recovered from a mutant murine cytomegalovirus (MCMV) BAC in which the M34 ORF had been deleted (BACDeltaM34). In contrast, an M34 mutant virus (RcM34), in which the M34 ORF was interrupted by transposon insertion at nt 44,827 of the Smith MCMV BAC, was attenuated in replication both in tissue culture and in SCID mice. Similarly, mutant virus Rc3'DeltaM34, in which the 3'-end was deleted from nt 44,724 to nt 45,647, produced similar replication kinetics in tissue culture to RcM34 while BAC5'DeltaM34, in which the 5'-end from nt 43,083 up to nt 44,896 was deleted, was non-viable like BACDeltaM34.

Ly49H+ NK cells migrate to and protect splenic white pulp stroma from murine cytomegalovirus infection.

In this study, we show that in the absence of a protective NK cell response, murine CMV causes destruction of splenic white and red pulp pulp areas in the first few days of infection. Destruction of T zone stroma is associated with almost complete loss of dendritic cells and T cells. We provide evidence that the virus replicates in red and white pulp stroma in vivo and in vitro.

Role of CD30 in B/T segregation in the spleen.

In this report, we identify an important function for CD30 signals in the effective segregation of B and T lymphocytes in the murine spleen, additional to the recognized requirement for lymphotoxin signals. We show that CD30 signals are not required for transcription or protein expression of homeostatic chemokines, but CD30-deficient mice display impaired B/T segregation. This defect correlates with defective expression as detected by Abs of the transmembrane mucin-type protein podoplanin on T zone stroma, although expression at other sites is normal.

Murine cytomegalovirus open reading frame m29.1 augments virus replication both in vitro and in vivo.

Murine cytomegalovirus mutant Rc29, with a premature stop codon mutation in the m29 open reading frame (ORF), produced no apparent phenotype in cell culture or following infection of BALB/c mice. In contrast, a similar mutant virus, Rc29.1, with a premature stop codon mutation in its m29.

Mutations in the temperature-sensitive murine cytomegalovirus (MCMV) mutants tsm5 and tsm30: a study of genes involved in immune evasion, DNA packaging and processing, and DNA replication.

A murine cytomegalovirus (MCMV) temperature-sensitive (ts) mutant, tsm5, of the K181 (Birmingham) strain, showed approximately 10-fold and approximately 10,000-fold reductions in yields at the permissive (33 degrees C) and non-permissive temperature (40 degrees C), respectively. It did not replicate to detectable levels in any tissue of 1-week-old Balb/c mice for up to 21 days following i.p.

Role of apoptosis and cytokines in influenza virus morbidity.

Influenza virus is a major human pathogen that causes epidemics and pandemics with increased morbidity and, especially in the elderly and those with pre-existing medical conditions, increased mortality. Influenza is characterised by respiratory symptoms and constitutional symptoms. Whilst knowledge of the mechanisms underlying host and tissue specificity has advanced considerably of late we still know relatively little about other aspects of influenza virus virulence.

Influenza A virus-induced apoptosis is a multifactorial process: exploiting reverse genetics to elucidate the role of influenza A virus proteins in virus-induced apoptosis.

Three influenza viruses, A/Puerto Rico/8/34-A/England/939/69 clone 7a (H3N2), A/Fiji/15899/83 (H1N1), and A/Victoria/3/75 (H3N2), induce different levels of apoptosis in vitro at equal moi; Clone 7a > A/Victoria > A/Fiji. Previous studies have shown that several viral proteins from clone 7a and A/Fiji, including PB2, NA, NS1, M1, and M2, induce apoptosis when expressed individually fused to the herpes simplex virus tegument protein, VP22. However, this did not reflect viral protein-protein-RNA interactions known to occur within infected cells.

The development of a modified human IFN-alpha2b linked to the Fc portion of human IgG1 as a novel potential therapeutic for the treatment of hepatitis C virus infection.

Interferon-alpha (IFN-alpha), in conjunction with ribavirin, is the current standard for the treatment of chronic hepatitis C virus (HCV) infection. This treatment requires frequent dosing, with a significant risk of the development of anti-IFN-alpha neutralizing antibodies that correlates with lack of efficacy or relapse. We have developed an IFN-alpha linked to the Fc region of human IgG1 for improved half-life and less frequent dosing.

Assessment of contraceptive vaccines based on recombinant mouse sperm protein PH20.

Mouse PH20 (mPH20), the mouse homologue to guinea pig hyaluronidase protein PH20 (gpPH20), was used to produce contraceptive vaccines that target both sexes of mice. Previously, immunization with a female gamete antigen (the zona pellucida subunit 3 protein) delivered in a recombinant murine cytomegalovirus (MCMV), or as a purified recombinant protein, has been shown to induce infertility in female mice. There is evidence, however, that sperm protein antigens could provide broader contraceptive coverage by affecting both males and females, and the most promising has been gpPH20 when tested in a guinea pig model.

Influenza A virus-induced apoptosis in bronchiolar epithelial (NCI-H292) cells limits pro-inflammatory cytokine release.

Infection of cells with influenza A virus results in cell death with apoptotic characteristics. Apoptosis is regarded as a non-inflammatory process. However, during influenza an inflammatory response occurs in the airway epithelium.

High-frequency interferon-gamma-secreting splenocytes specific for murine cytomegalovirus immediate-early-1 (IE-1) peptide 168YPHFMPTNL176 are insufficient to provide complete protection from viral challenge.

Infection of Balb/c mice with murine cytomegalovirus (MCMV) has been used extensively as a model system with which to study host mechanisms of immunity to cytomegaloviruses. In this model, the cytotoxic T-lymphocyte (CTL) response crucial for clearing infected cells is dominated by CTLs specific for the MCMV nonapeptide 168YPHFMPTNL176 encoded by the immediate-early 1 (IE-1) gene. The intradermal injection of plasmid pcDNA89 encoding IE-1 has been shown to offer some protection from viral challenge.

Immunisation of Balb/c mice with severely attenuated murine cytomegalovirus mutants induces protective cellular and humoral immunity.

Previously, we showed that two temperature-sensitive mutants of murine cytomegalovirus (tsm5 and tsm30) expressed immediate-early (IE-1), early (E-1), and late (gB) phase genes in the tissues of immunocompetent Balb/c mice, yet failed to produce infectious progeny virus in any tissue at any time at 1-21 days post-infection. Mice inoculated intraperitoneally with tsm5 became latently infected, but this latent virus could not be reactivated as an infectious virus after immunosuppression, although all three transcripts were produced. Immunocompetent mice infected with tsm30 did not become latently infected.

Oral administration of cyclopentane neuraminidase inhibitors protects ferrets against influenza virus infection.

Several cyclopentane inhibitors of influenza virus neuraminidase that have inhibitory activities in tissue culture similar to those of zanamivir and oseltamivir have recently been described. These new inhibitors have been examined for efficacy against a virulent H3N2 influenza virus when administered orally to infected ferrets. Preliminary studies indicated that oral administration of BCX-1923, BCX-1827, or BCX-1812 (RWJ-270201) at a dose of 5 or 25 mg/kg of body weight was active in ferrets in reducing respiratory and constitutional signs and symptoms, but these antivirals affected virus titers in the upper and lower respiratory tracts only marginally.