Jon D. Reuter

Complete Protection from Papillomavirus Challenge after a Single Vaccination with a Vesicular Stomatitis Virus Vector Expressing High Levels of L1 Protein

ABSTRACT We generated an attenuated, recombinant vesicular stomatitis virus (VSV) expressing high levels of the cottontail rabbit papillomavirus (CRPV) L1 protein from an upstream site in the VSV genome. Rabbits vaccinated once with this VSV-L1 recombinant produced high levels of anti-L1 antibody and were completely protected against papilloma formation after challenge with CRPV. In contrast, animals vaccinated only once with a VSV vector expressing lower levels of L1 from a downstream site in the VSV genome generated lower levels of L1 antibody and demonstrated only incomplete protection from papilloma formation after challenge. We conclude that the level of L1 protein expression is critical in generating complete immunity with a single-dose vaccine.

Enhanced Cytomegalovirus Infection of Developing Brain Independent of the Adaptive Immune System

ABSTRACT Cytomegalovirus (CMV) has been suggested as the most prevalent infectious agent causing neurological dysfunction in the developing brain; in contrast, CMV infections are rare in the adult brain. One explanation generally given for the developmental susceptibility to the virus is that the developing immune system is too immature to protect the central nervous system from viral infection, but as the immune system develops it can protect the brain. We suggest an alternate view: that developing brain cells are inherently more susceptible to CMV infection, independent of the immune system. We used a recombinant mouse CMV that leads to green fluorescent protein expression in infected cells. Control experiments demonstrated a high correlation between the number of cells detected with the viral GFP reporter gene and with immunocytochemical detection of the virus. After intracerebral inoculation, the number of CMV-infected cells in neonatal brains was many times greater than in mature control or mature immunodepressed SCID mice, and the mortality rate of neonates was substantially greater than SCID or control adults. Parallel experiments with live brain slices inoculated in vitro, done in the absence of the systemic immune system, generated similar data, with immature hippocampus, hypothalamus, cortex, striatum, and cerebellum showing substantially greater numbers of infected cells (100-fold) than found in adult slices in these same regions. Interestingly, in the cerebellar cortex, CMV-infected cells were more prevalent in the postmitotic Purkinje cell layer than in the mitotic granule cell layer, suggesting a selective infection of some cell types not dependent on cell division. Together, these data support the view that CMV has an intrinsic preference for infection of developing brain cells, independent, but not mutually exclusive, of the developmental status of the systemic immune system in controlling CMV infection.

Systemic Immune Deficiency Necessary for Cytomegalovirus Invasion of the Mature Brain

ABSTRACT Cytomegalovirus (CMV) is a significant opportunistic pathogen associated with AIDS and immunosuppressive therapy. Infection of the mature central nervous system (CNS) can cause significant pathology with associated neurological deficits, mental disorders, and cognitive impairment and may have potentially fatal consequences. Using genetically immunocompromised mice, we studied mechanisms of CMV invasion into, and behavior within, the CNS. Adult immunodeficient (nude and SCID) and control mice were peripherally infected with recombinant mouse CMV expressing a green fluorescent protein reporter gene. Control mice actively eliminated acute peripheral infection and were resistant to invasion of CMV into the brain. In contrast, virus infected brains of immunodeficient mice but only after a minimum of 21 days postinoculation. After inoculation, CMV was found in circulating leukocytes (MAC-3/CD45+) and in leukocytes within the brain, suggesting these cells as a possible source of CMV entry into the CNS. CNS infection was observed in many different cell types, including neurons, glial cells, meninges, ependymal cells, and cells of cerebral vessels. Infection foci progressively expanded locally to adjacent cells, resulting in meningitis, choroiditis, encephalitis, vasculitis, and necrosis; clear indication of axonal transport of CMV was not found. Regional distribution of CMV was unique in each brain, consisting of randomly distributed, unilateral foci. Testing whether CMV gained access to brain through nonspecific vascular disruption, vascular injections of a tracer molecule revealed no obvious disruption of the blood brain barrier in mice with CMV in the brain. Results indicate the importance of host adaptive immunity (particularly T cells) in controlling entry and dissemination of CMV into the brain and are consistent with the view that virus may be carried into the brain by circulating mononuclear cells that traffic through the blood brain barrier.

Optimization of cottontail rabbit papilloma virus challenge technique

Disease induced by Cottontail Rabbit Papilloma Virus (CRPV) scarification in domestic rabbits shares many attributes with disease induced by human papilloma virus (HPV). CRPV induces squamous papillomas in domestic rabbits, of which approximately 70% transform into invasive carcinomas. In advanced tumors, virus is often undetectable, and occasionally, some rabbits undergo spontaneous regression of papillomas. Techniques utilized to scarify rabbit skin are diverse, often labor intensive and time consuming with the possibility for significant variability. Using four unique infection techniques, resultant papilloma incidence, time to onset, and total papilloma volumes were compared to determine an optimal challenge method. Five rabbits were each infected with CRPV via a tattoo gun with and without ink, an intradermal injection, manual use of a tattoo needle, or a sterile blade followed by manual use of a tattoo needle. Papilloma formation was monitored weekly after inoculation for 6 weeks. CRPV papillomas began as pinpoint foci at 3 weeks post challenge and grew exponentially throughout the course of measurement. Individual foci coalesced rapidly to form larger papilloma aggregates. Although intradermal injection was well tolerated and easily performed, it was the worst method of papilloma production (2.2 mm(3) at 6 weeks). The best method, a sterile blade followed by manual use of a tattoo needle, produced significantly larger papillomas over all time periods (>1100 mm(3) at 6 weeks, P<0.01). Inoculation of CRPV using this method produces highly repeatable papillomas beginning 3 weeks post-infection.

CD4+ T-Cell Reconstitution Reduces Cytomegalovirus in the Immunocompromised Brain

ABSTRACT Cytomegalovirus (CMV) infection is the most common opportunistic infection of the central nervous system in patients with human immunodeficiency virus or AIDS or on immunosuppressive drug therapy. Despite medical management, infection may be refractory to treatment and continues to cause significant morbidity and mortality. We investigated adoptive transfer as an approach to treat and prevent neurotropic CMV infection in an adult immunodeficient mouse model. SCID mice were challenged with intracranial murine CMV (MCMV) and reconstituted with MCMV- or vesicular stomatitis virus (VSV)-sensitized splenocytes, T cells, or T-cell subsets. T cells labeled with vital dye or that constitutively generated green fluorescent protein (GFP) were identified in the brain as early as 3 days following peripheral transfer. Regardless of specificity, activated T cells localized to regions of the brain containing CMV, however, only those specific for CMV were effective at clearing virus. Reconstitution with unsorted MCMV-immune splenocytes, enriched T-cell fractions, or CD4+ cells significantly reduced virus levels in the brain within 7 days and also prevented clinical disease, in significant contrast with mice given VSV-immune unsorted splenocytes, MCMV-immune CD8+ T cells, and SCID control mice. Results suggest CMV-immune T cells (particularly CD4+) rapidly cross the blood-brain barrier, congregate at sites of specific CMV infection, and functionally eliminate acute CMV within the brain. In addition, when CMV-immune splenocytes were administered prior to a peripheral CMV challenge, CMV entry into the immunocompromised brain was prevented. Systemic adoptive transfer may be a rapid and effective approach to preventing CMV entrance into the brain and for reducing neurotropic infection.