Cell carriage, delivery, and selective replication of an oncolytic virus in tumor in patients

Oncolytic viruses, which preferentially lyse cancer cells and stimulate an antitumor immune response, represent a promising approach to the treatment of cancer. However, how they evade the antiviral immune response and their selective delivery to, and replication in, tumor over normal tissue has not been investigated in humans. Here, we treated patients with a single cycle of intravenous reovirus before planned surgery to resect colorectal cancer metastases in the liver. Tracking the viral genome in the circulation showed that reovirus could be detected in plasma and blood mononuclear, granulocyte, and platelet cell compartments after infusion. Despite the presence of neutralizing antibodies before viral infusion in all patients, replication-competent reovirus that retained cytotoxicity was recovered from blood cells but not plasma, suggesting that transport by cells could protect virus for potential delivery to tumors. Analysis of surgical specimens demonstrated greater, preferential expression of reovirus protein in malignant cells compared to either tumor stroma or surrounding normal liver tissue. There was evidence of viral factories within tumor, and recovery of replicating virus from tumor (but not normal liver) was achieved in all four patients from whom fresh tissue was available. Hence, reovirus could be protected from neutralizing antibodies after systemic administration by immune cell carriage, which delivered reovirus to tumor. These findings suggest new preclinical and clinical scheduling and treatment combination strategies to enhance in vivo immune evasion and effective intravenous delivery of oncolytic viruses to patients in vivo.     

Pathogenicity of Ife virus for Swiss albino mice

Pathogenicity of Ife virus was studied in Swiss albino mice following four inoculation routes. Mice of all ages survived oral infection without seroconversion; subcutaneous (i.c.) and intraperitoneal (i.p.) infections lead to low titre antibody production. Only suckling mice (1-5 days old) succumbed to intracerebral (i.c.) inoculation with infectivity titres which decreased by age and average survival time (AST) increasing with age. Following i.c. inoculation to suckling mice, the brain infectivity titres increased progressively by days post-infection (p.i.). Virus was not recovered from the lungs and kidney but in low titre it was obtained from the liver, spleen, heart and blood at different days p.i. All organs examined showed evidence of complement fixing and immunofluorescent Ife virus antigen. No gross lesions were observed. The histopathological lesions were limited to the brain.     

Growth characteristics and immunocytochemical studies of reovirus type 2 in a line of human amnion cells

Summary The intracellular development of reovirus type 2 and its interaction with an established human amnion cell line (RA) have been investigated. Adsorption studies indicate that maximal adsorption of the virus to the RA cell was attained within 75–90 minutes after exposure. Single cycle growth studies revealed an eclipse period of 9 hours, and maximal yields were reached at 36 hours post-infection (p.i.). In comparison, the eclipse period for reovirus type 3 in the same cell line was 6 hours, with maximal yields attained at approximately 26 hours p.i. Immunofluorescent and cytochemical (acridine orange) examination of virus-induced alterations in the cell during a single growth cycle of reovirus type 2 revealed the following: a) appearance of virus antigen in the perinuclear region as early as 4 hours p.i. and a maximum number of cells containing antigen by 10 hours p.i.; b) appearance of orthochromatically green-staining perinuclear inclusions at 6 hours p.i. These inclusions were resistant to digestion by ribonuclease and deoxyribonuclease. Metachromatically red-staining inclusions sensitive to ribonuclease were observed to appear in a few cells late in the infectious cycle.This investigation was supported by grant AI 04685 from the National Institute of Allergy and Infectious Diseases of the National Institutes of Health.NIH Trainee 5 TI AI 243-02.     

Immune response to foot-and-mouth disease virus in a murine experimental model: effective thymus-independent primary and secondary reaction.

The immune response against foot-and-mouth disease virus (FMDV) was studied in a murine model. In untreated control mice, the inoculation of 10,000 suckling mouse 50% lethal doses of Ol Campos FMDV i.p. was followed by a burst of viraemia that disappeared in less than 4 days, i.e. when the neutralizing antibodies (NAb) reached titres above one neutralizing unit. In mice treated with cyclophosphamide, the curves of viraemia and NAb were significantly delayed. Nu/nu mice injected with FMDV had curves of viraemia and NAb identical to those of their nu/t littermates. We then studied the secondary (memory) immune reaction in the same model. In order to investigate which preimmunized cells participate in the elimination of actively replicating FMDV, mice were irradiated, then infected with FMDV, and 24 hr later repopulated with cells obtained from either donor mice that had been previously immunized by infection with live virus, or non-infected controls. The transfer of control (non-immunized) lymphoid cells was unable to eliminate the viraemia in recipient animals at times significantly different from those observed with irradiated recipients receiving no cells, while repopulation of recipients with 10(8) immune lymphoid cells (obtained from pooled thymus, blood, peritoneal exudate, spleen and lymph nodes of preinfected donor mice) led to undetectable titres of viraemia at Day 5 post-infection (p.i.). High doses of thymus cells were totally inactive, while a few as 10(7) donor spleen cells were able to abort viraemia at 6 days p.i. When enriched preparations of B or T spleen cells were adoptively transferred, only B cells were able to abort viraemia in irradiated recipients. It is concluded that, in the murine model of FMDV infection, B cells are mainly responsible for primary response and short-term immunological memory. In both cases the protective immune reaction is T-independent.     

Infection of mouse liver by human adenovirus type 5.

CBA mice, inoculated intravenously with large doses of adenovirus type 5, showed raised levels of serum aspartate aminotransferase (SAAT; EC 2.6.I.I) and died within a few days from histologically demonstrable hepatic necrosis. After inoculation of I LD50, virus was rapidly taken up by the tissues where infectivity then declined greatly. Organ titres then increased about 100-fold by 48 h p.i. but, in the liver, which showed intranuclear inclusion bodies, and by electron microscopy, scattered intranuclear and intracytoplasmic adenovirions, the increase was 10000- to 100000-fold. P antigen was detected by single radial diffusion in liver extracts, and by immunofluorescence in 80% of liver cells at 36 h p.i. Hexon, penton base and fibre antigens appeared later and in fewer cells. The maximum amount of hexon, of demonstrable type 5 specificity, was shown by radioimmunoassay to be equivalent to up to 5 x 1011 whole adenovirions/g liver. It is concluded that human adenovirus type 5 undergoes an abortive but lytic infection in most liver cells but that replication may proceed to completion in a few.     

Studies on pathogenesis of fowl pox: virological study

One-month-old WLH chickens were inoculated with a field isolate of fowl pox virus (FPV) by intradermal (i.d.) and intratracheal (i.t.) routes. In intradermally infected chickens, the virus in titrable amounts was first detected in the skin at the inoculation site on day 2 and in lungs on day 4 followed by viraemia on the day 5 post-infection (p.i.). Subsequently the virus was recovered from liver, spleen, kidney and brain, but not from the heart. The chickens infected by i.t. route showed an almost similar outcome with minor differences as the virus was first demonstrated in the lungs on day 2 p.i., viraemia occurred on day 4 p.i. Initiation of pocks at the inoculation site in i.d. infected birds was observed on days 3 to 4 p.i., generalized cutaneous pock lesions appeared from 7 to 8 days p.i.     

Antiserum-induced neutropenia in the rat: characterization of a rabbit anti-rat neutrophil serum.

A heterologous rabbit anti-rat neutrophil serum (ANS) based on peptone-stimulated peritoneal exudate neutrophils (PMNLs) from Sprague Dawley rats was prepared. Leucoagglutination and indirect immunofluorescence assays revealed high titres of antibodies to rat PMNLs (1/2560), lower titre of antibodies to rat lymphocytes (1/160) and a very low titre against rat platelets (1/20). ANS given intravenously (i.v.) to rats in doses of up to 42 mg of protein/kg b.w. caused transient neutropenia, lasting about 10 min after administration, and thrombocytopenia, lasting about 5 min. Two minutes after an i.v. injection of 21 mg of ANS/kg b.w. there was profound uptake of 51Cr-labelled PMNLs in the lung, increased release of 51Cr to plasma, an increased amount of 51Cr in the spleen and consumption of greater than 98% of total complement (CH50). Two hours later there was high activity of 51Cr in the plasma, spleen and liver, while lung radioactivity had decreased to below baseline and CH50 had recovered to 55% of baseline. An intraperitoneal (i.p.) injection of ANS was followed by prolonged neutropenia with a maximum after 12 h. Simultaneously peripheral mononuclear cells slightly decreased. There was no change in the number of peripheral platelets in the blood or in the plasma concentration of fibrinogen, alpha 2-antiplasmin, plasminogen or plasminogen activators. Intraperitoneal administration of ANS did not affect CH50. It was concluded that the raised ANS had good specificity against rat PMNLs and was able to induce prolonged neutropenia after i.p. injection without affecting the complement of fibrinolytic system.     

Stability of infectious HIV in clinical samples and isolation from small volumes of whole blood.

AIMS--To evaluate the stability of infectious HIV in clinical samples and the efficiency of isolating it from small volumes of whole blood. METHODS--Titres of infectious HIV were measured in peripheral blood mononuclear cells and plasma 2, 24, and 48 hours after peripheral blood had been collected from 10 HIV positive adult volunteers. Volumes of whole blood (1 ml to 5 microliters), collected from a further five volunteers, were used to determine the minimum volume from which HIV could be isolated. Infectious HIV was isolated by co-culture with phytohaemagglutinin stimulated umbilical cord mononuclear cells. RESULTS--Geometric mean titres of infectious HIV seemed to be more stable in peripheral blood mononuclear cells than in plasma. HIV was recovered from all 10 peripheral blood mononuclear cell samples during the 48 hours after sample collection, but from only four plasma samples. HIV could occasionally be isolated from 5 microliters of whole blood and reliably from 200 microliters. CONCLUSIONS--HIV can be isolated from peripheral blood mononuclear cells and plasma for up to 48 hours after sample collection. Isolation of HIV from small volumes of whole blood has applications for the diagnosis and management, of HIV positive children.     

Study of hepatitis C virus leukotropism by characterization of viral quasispecies in the liver transplantation setting

Besides hepatocytes, representing the main replication site of hepatitis C virus, peripheral blood mononuclear cells also represent a crucial target for viral infection. Hepatitis C virus compartmentalization (i.e., non-random distribution) of viral variants between plasma and peripheral blood mononuclear cells, more frequently observed in liver transplant patients compared to non-transplanted patients, makes liver transplantation an interesting model for the analysis of hepatitis C leukotropism. This article aims to present, firstly, in clinical and biological features arguing favour of hepatitis C virus infection leukotropism and, secondly, to review current knowledge about compartmentalization between plasma and peripheral blood mononuclear cells, especially in the liver transplantation setting.     

The core-specific precursor T cell response is directed to the N-terminal and central parts of the protein and positively correlates to the viral load in chronically HCV-infected patients

The cellular immune response to hepatitis C virus (HCV) plays a critical role in determining the clearance or persistence of HCV. Moreover, in chronic HCV infection, these responses that are insufficient to eradicate virus completely may cause liver injury. In this study, the memory T cells responses specific to the core protein were measured by interferon-gamma Elispot assay after in vitro stimulation of peripheral blood mononuclear lymphocytes from chronically infected subjects. Ten out of the 22 patients studied (45%) present a core-specific response with a preferential recognition of the N-terminal and central parts. There was no relationship between T cell responses and the parameters of disease evolution as determined by ALT (serum alanine transaminase levels), and histologic hepatic damage (Metavir score A and F), but there was a positive relationship between the presence of a core-specific T cell responses and the viraemia.     

Viraemia and antibody response of the mallard (Anas platyrhynchos) to infection with tick-borne encephalitis virus

The course and intensity of viraemia after experimental infection with a TBE virus was studied in mallards of varying age and weight. Although virus titres in the blood can range from log_10^2·65 to log_10^4·85, in general these titres are believed to be of sufficient magnitude to infect ticks with the virus.Neutralizing antibodies in the serum of ducks are long-lasting and of sufficient titre to prevent a second viraemia. That mallards can play a role in the epidemiology of TBE virus is likely. Although some ducklings died in unexplained circumstances, none of the infected mallards showed overt disease.     

Herpes simplex virus type 1 and 2 in the adrenal glands: Replication and histopathology

Summary The adrenal glands were shown to be the most severely infected organs in the early phase of HSV-1 infections (up to 10 days p. i.) after i. p. infections in mice. Virus could be isolated from the adrenal glands as early as one hour after infection with pathogenic and apathogenic strains. Infection of the adrenal glands is a result of viremia. The content of HSV-1 (5 strains) was much higher in the adrenals than in spleen and liver. It peaked at 3–4 days p. i. compared to 1–2 days in spleen and liver. Only strain 17 syn⁺ produced low tissue titres in the adrenal glands.Morphologic alterations by HSV-1 infections commenced with distinct foci 2 days after infection in the zona fasciculata, detected immunohistochemically by HSV-specific peroxidase-staining. Necrotic cells could be observed. The foci became confluent until day 4 and remained in this status up to day 7 p. i. During infection immunocompetent cells (macrophages, granulocytes, many T-helper — but only few T-cytotoxic/suppressor lymphocytes) could be observed. On day 10 p. i. the viral antigen had been completely eliminated.In contrast, intraperitoneal infections with 5 strains of HSV-2 resulted in infection of the adrenal glands only to a low degree. The titer of virus was low (exception: strain HG 52). This correlates well with the type of disease produced by either HSV-1 or 2. By comparing the replication of different strains of HSV-1 and 2, three types of tropism after i. p. infection of mice can be distinguished.With 7 FiguresIn partial fulfillment of his M. D.'s degree.     

Liver disease in rhesus monkeys infected with simian immunodeficiency virus.

Rhesus monkeys infected with simian immunodeficiency virus (SIV) develop a syndrome very similar to patients with acquired immune deficiency (AIDS), including liver disease. This prospective study was undertaken to define the pathology, course, and pathogenesis of liver disease in 20 rhesus monkeys (Macaca mulatta) after intravenous inoculation with the standardized isolate SIV/DeltaB670. Tissue samples from liver and gallbladder between 2 and 24 weeks after inoculation were examined histologically and immunohistochemically for SIV gag protein p26, and by in situ hybridization with an SIV riboprobe. Histologically there was infiltration of portal tracts and around hepatic veins and venules by mononuclear inflammatory cells, focal bile duct damage, proliferation of bile ductules, and focal lobular inflammation as early as 2 weeks after infection. The severity and extent of these lesions were graded semiquantitatively and showed that bile duct damage and hepatic venulitis were the most significant changes. Simian immunodeficiency virus gag protein p26 and SIV RNA were detected in scattered mononuclear cells in portal tracts and sinusoids, but not in hepatocytes or bile duct epithelial cells. The data indicate that the liver is involved early during the course of SIV infection, followed by persistent changes until the terminal stage of the disease. Our findings suggest that the liver damage in SIV-infected rhesus monkeys is similar to the changes observed previously in AIDS patients.     

Feline immunodeficiency virus infection: plasma,but not peripheral blood mononuclear cell virus titer is influenced by zidovudine and cyclosporine

Summary The plasma and peripheral blood mononuclear cell (PBMC) titer of feline immunodeficiency virus (FIV) in experimentally infected cats was assessed following administration of either zidovudine or cyclosporine. Treatments were begun 24 h post infection (p.i.) and continued for 4 weeks. Zidovudine treatment did not prevent establishment of infection with FIV, but plasma virus titers were significantly lower than controls at 2 weeks p.i. This reduction of plasma virus titer by zidovudine was not maintained at subsequent sampling times. Similarly, cyclosporine treatment initially lowered plasma virus titers at 2 weeks p.i., but at 4 weeks p.i. the plasma virus titers in cyclosporine-treated cats were significantly higher than in the untreated group. In the untreated group, plasma virus titers declined rapidly to an undetectable level by 14 weeks p.i. Neither zidovudine or cyclosporine treatment significantly influenced the titer of FIV in PBMCs. In all groups (untreated, zidovudine and cyclosporine) the titers in PBMC were high for the duration of the experiment. The decline in plasma virus titers in immunocompetent cats combined with the effect of cyclosporine on plasma titers strongly suggests that the immune system plays a major role in clearing FIV from plasma. In contrast, it appears that the immune response has little impact on PBMC virus titers. This shows that for complete assessment of antiviral agents, both cell-free and cell-associated virus titers must be examined. We also suggest that the limitation of viral titers in PBMC may be of critical importance in the control of lentiviral infection.     

Pathology and virus dispersion in cynomolgus monkeys experimentally infected with severe acute respiratory syndrome coronavirus via different inoculation routes

Severe acute respiratory syndrome-associated coronavirus (SARS-CoV) causes SARS. The pathogenic mechanisms of SARS-CoV remain poorly understood. Six cynomolgus monkeys were inoculated with the HKU39849 isolate of SARS-CoV via four routes. After intranasal inoculation, the virus was isolated from respiratory swabs on days 2-7 postinoculation (p.i.) and virus genome was detected in intestinal tissues on day 7 p.i. Virus was not detected after intragastric inoculation. After intravenous inoculation, infectious virus was isolated from rectal swabs, and virus antigen was detected in intestinal cells on day 14 p.i. After intratracheal (i.t.) inoculation, virus antigen-positive alveolar cells and macrophages were found in lung and infectious virus was detected in lymphoid and intestinal tissues. The peribronchial lymph nodes showed evidence of an immune response. Lung tissue and/or fluid and/or the peribronchial lymph node of the intratracheally inoculated animals had high TNF-alpha, IL-8 and IL-12 levels. SARS lung lesions are only generated in monkeys by i.t. inoculation. The virus appears to spread into and perhaps via the intestinal and lymphatic systems. It has been suggested previously that viraemia may cause intestinal infections in SARS patients.     

Development of a rapid quantitative assay for HIV-1 plasma infectious viraemia-culture-PCR (CPID)

A simple and rapid assay for the quantification of infectious HIV-1 in plasma was developed using short-term culture and DNA PCR. This method, called culture PCR, allows detection and quantification of infectious HIV-1 viraemia within 48 hours, and measures the number of infectious cell-free HIV-1 particles, expressed as culture PCR infectious doses (CPID/ml). 42 HIV infected subjects were assessed by this method. The titres obtained by CPID closely correlated with CD4⁺ count and clinical status. CPID titres had significant correlation with infectious virus titre determined by conventional limiting dilution tissue-culture methods. This culture-PCR technique permits rapid assessment of infectious plasma viraemia, and is comparable to longer culture based assay methods. © 1994 Wiley-Liss, Inc.     

Reinfection and reactivation of equine herpesvirus-1 in the mouse

Summary Balb/c mice were inoculated with equine herpesvirsus-1 (EHV-1) by the intranasal (i.n.) route. Mice developed respiratory signs; virus replication occurred in the respiratory tract and viraemia was detected; some mice died. Recovered mice were given a second inoculation with the same strain 5 months later. Following the second infection no mice died, however, virus replication was again observed in the respiratory tract and viraemia was detected once more. Administration of an antiviral agent during the acute infection prevented mice from developing severe clinical signs and all survived. These mice, and some that had survived an acute infection without chemotherapy, were given a variety of stimuli, for example X-irradiation or corticosteroid injection. Reappearance of infectious virus was detected in approx. 1/3 animals in either the respiratory tract or blood. We speculate on the possible sites of latency in the model.     

Egg transmission of avian reoviruses in chickens: Comparison of a trypsin-sensitive and a trypsin-resistant strain

Two groups of commercial Light Sussex hens with no cultural evidence of reovirus infection and very low titres of neutralising antibodies were mated with cockerels from 17 weeks of age. At 27 weeks of age the birds were separated into three groups, and were inoculated intranasally and intravenously with avian reovirus strain R2 which is resistant to trypsin, with strain TR1 which is sensitive to the enzyme or sham-inoculated. Of the eggs laid by the hens infected with strain R2, 13/29 infertile eggs and embryos which fails to hatch were positive for virus, as were 6/70 hatched chicks. Despite this, virus was never isolated from cloacal swabs from the hens. Virus-infected eggs were laid between days 5 to 17 post inoculation (p.i.). Virus was isolated from the liver of all six R2 virus-positive chicks, from the hock joint of four and from the intestine of three. In contrast, for the group infected with the trypsin-sensitive virus TR1, of 120 eggs laid in the 5-week period, virus was isolated once only, from a chick hatched from an egg laid 7 days p.i. This infected chick was one of 83 which hatched and virus was found only in the joint. In a further experiment, two groups of mature SPF hens were inoculated with the reoviruses as above. Cloacal swabs and tissue examination showed greater virus excretion and tissue distribution of R2 than TR1. These results helped to explain the much higher egg transmission rate of R2 than TR1. However, the rate of vertical transmission of chicken reoviruses in nature, where the infectious dose would normally be lower than given here, is likely to be low.     

The effect of virus dose on the development of Marek's disease in two strains of chickens

The effect of inoculating different doses of Marek's disease virus on the consequent lymphocyte-associated viraemia titres, survival time and mortality was studied in two strains of chickens, one highly susceptible (a strain of Rhode Island Red) and one moderately resistant (a strain of Light Sussex) to Marek's disease. In both strains an increase in the infecting dose of virus increased the ensuing viraemia, and there was an inverse relationship between virus dose and survival time. There was a negative correlation between viraemia titres and survival time. The modulation of infecting dose had no effect on overall mortality among the susceptible, Rhode Island Red strain, chickens, but did influence the mortality of the Light Sussex strain chickens. It was suggested that the outcome of infection within an individual may be determined by a rather subtle interaction between the infection and transformation of a limiting number of target cells and an immune response directed against such infected or transformed cells, and that in different breeds of chickens these two factors may differ in importance.