HIV-specific T lymphocyte immunity in mice immunized with a recombinant vaccinia virus

Infection by the human immunodeficiency virus (HIV) induces T cell immunity in humans, chimpanzees and macaques. The protective value of this immune response is not clear. We have consequently developed a murine experimental system to study HIV-specific CD4 and CD8 T lymphocyte immunity in vitro and in vivo. BALB/c, DBA/2 and C3H/He mice were immunized with vaccinia virus (VV) recombinant VV-11.39 which expresses the gp160 glycoprotein of HIV-1. Primary and secondary cytotoxic T lymphocyte response to HIV were detected with histocompatible mouse target cells transfected with the HIV-1 env gene. Killer cells were positive for the Thy-1 and Ly-2 (CD8) T cell markers, and were restricted by class I H-2 histocompatibility antigens. Immunological memory specific for HIV-1 envelope antigens was clearly induced by vaccination with VV-11.39:spleen cells from mice vaccinated 4 weeks or more prior to assay generated CD4 and CD8 T lymphocyte responses following stimulation in vitro with HIV envelope antigens. The intensity of these responses increased with consecutive vaccinations, indicating that HIV-specific precursor T cell pools were progressively amplified. Finally, DBA/2 mice vaccinated with VV-11.39 developed protective immunity against a syngeneic tumor which expresses HIV-1 env antigens, leading to accelerated tumor rejection and increased survival.     

Neutrophil and remnant clearance in immunity and inflammation

Neutrophil-centred inflammation and flawed clearance of neutrophils cause and exuberate multiple pathological conditions. These most abundant leukocytes exhibit very high daily turnover in steady-state and stress conditions. Various armours including oxidative burst, NETs and proteases function against pathogens, but also dispose neutrophils to spawn pro-inflammatory responses. Neutrophils undergo death through different pathways upon ageing, infection, executing the intruder's elimination. These include non-lytic apoptosis and other lytic deaths including NETosis, necroptosis and pyroptosis with distinct disintegration of the cellular membrane. This causes release and presence of different intracellular cytotoxic, and tissue-damaging content as cell remnants in the extracellular environment. The apoptotic cells and apoptotic bodies get cleared with non-inflammatory outcomes, while lytic deaths associated remnants including histones and cell-free DNA cause pro-inflammatory responses. Indeed, the enhanced frequencies of neutrophil-associated proteases, cell-free DNA and autoantibodies in diverse pathologies including sepsis, asthma, lupus and rheumatoid arthritis, imply disturbed neutrophil resolution programmes in inflammatory and autoimmune diseases. Thus, the clearance mechanisms of neutrophils and associated remnants are vital for therapeutics. Though studies focused on clearance mechanisms of senescent or apoptotic neutrophils so far generated a good understanding of the same, clearance of neutrophils undergoing distinct lytic deaths, including NETs, are being the subjects of intense investigations. Here, in this review, we are providing the current updates in the clearance mechanisms of apoptotic neutrophils and focusing on not so well-defined recognition, uptake and degradation of neutrophils undergoing lytic death and associated remnants that may provide new therapeutic approaches in inflammation and autoimmunity.     

Treatment with IL2/vaccinia recombinant virus leads to serologic, histologic and phenotypic normalization of autoimmune MRL/lpr-lpr mice.

We have analyzed the effect of IL2 administered in vivo on both the lymphoproliferation and autoimmune disease progression of MRL/lpr mice. Human IL2 was delivered by infecting MRL/lpr mice with vaccinia virus recombinants at different stages of lpr disease. The results reported here showed that treatment of lpr mice with IL2 mediated: (1) restored normal thymic differentiation illustrated by an expansion of the double positive population accompanied by increased numbers of mature thymocytes; (2) depletion of the peripheral CD3+ CD4- CD8- (DN) T-cell population; (3) normalization in the pattern of TcRV beta gene expression displayed by mature T cells; (4) decreased urine-protein levels and immune complex deposition in the kidney, with a resultant absence of glomerulonephritis; and (5) an increased longevity (from 195 to more than 400 days). We speculate that the dramatic reduction in the abnormally expanded CD3+ DN T-cell population following IL2 therapy might be directly related to the amelioration and/or prevention of autoimmune disease in these mice. Collectively, these results suggest that diseases showing a selective expansion of DN cells should be envisaged as possible targets for the treatment described here.     

Presenilin 1 familial Alzheimer's disease mutation leads to defective associative learning and impaired adult neurogenesis

Alzheimer's disease is a learning and memory disorder pathologically characterized by the deposition of beta-amyloid plaques and loss of neurons and synapses in affected areas of the brain. Mutations in presenilin 1 (PS1) lead to the most aggressive form of familial Alzheimer's disease (FAD), and are associated with accelerated plaque deposition. However, since the function of PS1 is pleiotropic, we reasoned that the FAD mutations may alter multiple PS1-mediated pathways, and the combination of which may account for the early onset nature of the disease phenotype. Using the PS1M146V knockin mice in which the M146V mutation was incorporated into the endogenous mouse PS1 gene, we report here that the FAD mutation results in impaired hippocampus-dependent associative learning, as measured by a contextual fear conditioning paradigm, at 3 months of age. This is correlated with reduced adult neurogenesis in the dentate gyrus. However, short-term and long-term synaptic plasticity in both area CA1 and dentate gyrus are not affected. Our results suggest that impaired adult neurogenesis may contribute to the memory deficit associated with FAD.     

Immune response in vaccinia virus infected mice treated with N,N'-bis/methylisatin-beta-thiosemicarbazone/-2-methylpiperazine

The effect of the compound N,N'-bis/methylisatin-beta-thiosemicarbazone/-2-methylpiperazine (bis-MIBTP) on immune response in BALB/c and Swiss mice have been studied in the course of vaccinia virus infection. Humoral response tested by neutralization and hemagglutination inhibiting antibodies was similar in compound-treated mice to this of untreated mice. Cell-mediated immune response, examined by spleen lymphocytes migration inhibition test, has been delayed or temporally depressed in bis-MIBTP treated mice as compared with the control group. High protective activity of the compound in vaccinia infected mice in spite of impairment of cellular immunity may indicate that antibodies have played an important role in recovery process from vaccinia infection.     

Dengue virus infection and immune response in humanized RAG2(-/-)gamma(c)(-/-) (RAG-hu) mice

Dengue viral (DENV) pathogenesis and vaccine studies are hampered by the lack of an ideal animal model mimicking human disease and eliciting an adaptive human immune response. Although currently available animal models have been very useful in dissecting some key aspects of disease pathogenesis, a major limitation with these is the lack of a human immune response. In this study, we sought to overcome this difficulty by utilizing a novel mouse model that permits multi-lineage human hematopoiesis and immune response following transplantation with human hematopoietic stem cells. To generate immunocompetent humanized mice, neonatal RAG2(-/-)gamma(c)(-/-) mice were xenografted with human CD34+ hematopoietic stem cells, resulting in de novo development of major functional cells of the human adaptive immune system. To evaluate susceptibility to dengue viral infection, humanized mice were challenged with DEN-2 serotype. Viremia lasting up to 3 weeks was detected in infected mice with viral titers reaching up to 10(6.3) RNA copies/ml. Fever characteristic of dengue was also noted in infected mice. Presence of human anti-dengue antibodies was evaluated using an antibody capture ELISA. Anti-dengue IgM was first detected by 2 weeks post-infection followed by IgG at 6 weeks. Sera from some of the infected mice were also found to be capable of dengue virus neutralization. Infected mouse sera showed reactivity with the viral envelope and capsid proteins in immunoprecipitation assay. These results demonstrate for the first time that humanized mice are capable of dengue viral primary human immune responses thus paving the way for new dengue immunopathogenesis and vaccine studies.     

Genetic control of parasite clearance leads to resistance to Plasmodium berghei ANKA infection and confers immunity

Unprecedented cure after infection with the lethal Plasmodium berghei ANKA was observed in an F2 progeny generated by intercrossing the wild-derived WLA and the laboratory C57BL/6 mouse strains. Resistant mice were able to clear parasitaemia and establish immunity. The observed resistance was disclosed as a combinatorial effect of genetic factors derived from the two parental strains. Genetic mapping of survival time showed that the WLA allele at a locus on chromosome 1 (colocalizing with Berghei resistance 1 (Berr1), a locus associated with resistance to experimental cerebral malaria) increases the probability to resist early death. Also, the C57Bl/6 allele at a novel locus on chromosome 9 (Berr3) confers overall resistance to this lethal Plasmodium infection. This report underlines the value of using wild-derived mouse strains to identify novel genetic factors in the aetiology of disease phenotypes, and provides a unique model for studying parasite clearance and immunity associated with malaria.     

Attenuated deletion mutants of vaccinia virus lacking the vaccinia growth factor are defective in replication in vivo

Understanding the molecular basis of virulence in poxvirus is of great importance for the development of recombinant vaccines using vaccinia virus as a vector. We have previously described mutants of vaccinia virus with deletions ranging from 20 to 21 kb at the left terminus and with attenuated phenotype. The virulence of these mutants was studied, using different routes of inoculation, for protection from wild-type challenge in mice and for replication in vivo. Regardless of the route of inoculation, the LD50 of the deletion mutants is at least 1000-fold higher than that of the wild-type. Results from protection experiments using viable and ultraviolet-inactivated viruses, and from determination of infectivity in different organs, indicated that the mutants were unable to replicate in vivo. Southern blot hybridization of viral DNA with pSC16, a plasmid containing the vaccinia growth factor (VGF) gene, revealed that in the IHD-W strain of vaccinia virus this gene is localized at the left terminus exclusively and that the mutants lack this gene. The results suggest that absence of the VGF gene is correlated with inability to replicate in vivo and decreased virulence.     

Absence of oropharyngeal vaccinia virus after vaccinia (smallpox) vaccination.

BACKGROUND: With the resumption of the vaccinia (smallpox) vaccination, questions regarding transmission risk prompted this study to determine whether vaccinia virus could be detected in the oropharynx of adults recently vaccinated with vaccinia (smallpox) vaccine. German, Russian, and American studies on the oropharyngeal presence of vaccinia virus revealed conflicting results in different age groups. OBJECTIVE: To measure vaccinia viral particle or antigen presence in the oropharynx of adult health care workers after vaccination with vaccinia (smallpox) vaccine using viral culture and high-sensitivity assays (polymerase chain reaction [PCR] and electrochemiluminescence) and to determine whether there is an association between the presence of vaccinia virus and adverse reactions. METHODS: A total of 155 adults (primary vaccinees and revaccinees) were enrolled for 1 baseline and 5 subsequent throat swabs. The swabs were evaluated using viral culture, PCR, and electrochemiluminescence. RESULTS: Of the 155 participants, 144 had more than 2 throat swabs in the 2 weeks after vaccination. Of the 801 specimens evaluated, there were no positive results by culture, PCR, or electrochemiluminescence except in the control samples (n = 6), which were positive by all 3 methods. CONCLUSIONS: Based on the absence of detectable vaccinia virus in this study population, one can be 95% certain that the true rate of vaccinia virus in the oropharynx of adults during the 2 weeks after vaccination with vaccinia (smallpox) vaccine is 0% to 3.3%. These data should be reassuring to the medical community and support the Advisory Committee on Immunization Practice guidelines that respiratory precautions are not necessary after vaccinia (smallpox) vaccination in healthy adults.     

The nonreplicating smallpox candidate vaccines defective vaccinia Lister (dVV-L) and modified vaccinia Ankara (MVA) elicit robust long-term protection

Current smallpox vaccines are live vaccinia viruses that replicate in the vaccinee inducing immunity against the deadly disease smallpox. Replication resulting in virus spread within the host, however, is the major cause of severe postvaccinal adverse events. Therefore, attenuated strains such as modified vaccinia Ankara (MVA) or LC16m8 are candidates as next generation vaccines. These strains are usually grown in primary cells in which mass production is difficult and have an unknown protective potential in humans. Proven vaccine strains of defined origin and modern production techniques are therefore desirable. In this study, defective vaccinia virus (dVV) lacking a gene essential for replication (derived from the Lister vaccine in a complementing cell line) was compared with the Wyeth smallpox vaccine strain and with MVA in mouse animal models using cowpox and ectromelia virus challenge. Similar to MVA, prime-boost immunizations with defective vaccinia induced robust long-term immunity, suggesting it as a promising next generation smallpox vaccine.     

In vitro assessment of cellular immunity to vaccinia virus: contribution of lymphocytes and macrophages

The possible contributions of lymphocytes and macrophages to immunity to vaccinia virus were examined in vitro by determination of antigen-specific inhibition of migration of macrophages and the replication of the virus in purified macrophages. Lymphocytes from various anatomic sites of vaccinia virus-immune rabbits mixed with macrophages from nonimmune rabbits resulted in antigen-specific inhibition of macrophage migration; purified lymphocytes of peritoneal exudates were found to be the most potent inhibitors. Macrophages from peritoneal exudates of immune rabbits purified to contain less than 1% lymphocytes were sensitive to inhibition of migration by vaccinia antigens. Virus grew in macrophages from nonimmune rabbits, but failed to replicate in macrophages from the peritoneal cavity of immune rabbits. Alveolar maracrophages from immune rabbits were not inhibited from migrating in the presence of the viral antigen, and the virus replicated in these cells. The data suggest, but do not prove, that cellular immunity to vaccinia virus in rabbits may be mediated both by lymphocytes and by macrophages.     

Cell-mediated immunity to Sendai virus infection in mice.

The development of a cell-mediated immune response to Sendai virus infection in mice was examined by the use of a 51Cr release assay of cytotoxicity. A low level of "background cytotoxicity" to Sendai virus-infected L cells was found in the spleens of uninfected CBA mice. Spleen cells from Sendai-infected mice showed an elevated level of cytotoxicity against these target cells for a period of 5 weeks, commencing 4 days after infection of the mice. A more transient response was observed in the spleens of mice infected with a serologically distinct virus, the Kunz strain of influenza. This cross-reacting, cell-mediated immune response was intermediate between that observed in unsensitized and Sendai-sensitized spleen cells. The relevance of these cell-mediated immune responses to respiratory tract virus infections is discussed.     

Macrophage antitumor activity: impaired responsiveness to interferon-gamma of macrophages from genetically defective mice

Macrophages (M phi) from the genetically defective mouse strains C3H/HeJ, A/J and P/J were unable to develop high levels of antitumor activities when stimulated either with immune recombinant interferon gamma (IFN-gamma) or with nonimmune IFN-alpha and IFN-beta, as compared to M phi or normal C3H/HeN mice. IFN-gamma appeared to be an exceptionally good activator of C3H/HeN M phi, as it could induce tumoricidal capacities 3000 times more efficiently than nonimmune IFN. The high efficiency of IFN-gamma as M phi activator was indeed confirmed on defective M phi. In fact, at high doses IFN-gamma could also induce significant levels of both cytolytic and cytostatic activity in M phi of A/J and P/J mice, although it could not increase cytotoxic activities of C3H/HeJ M phi.     

Investigation of vaccinia virus DNA replication employing a conditional lethal mutant defective in DNA

After infection of L cells with the DNA-defective temperature-sensitive (ts) mutant 6389 of vaccinia virus, [3H]thymidine incorporation into cytoplasmic DNA is inhibited at 39 degrees, but resumes upon shiftdown to 32 degrees, the permissive temperature. Following a 30-min lag period DNA synthesis is linear and contingent upon continuous protein synthesis. Sedimentation analysis of nascent DNA labeled during 10 to 60-min pulses revealed that the mutant molecules are produced at a slower rate, but are approximately the same size as those of wild-type vaccinia, synthesized under the same circumstances. During more prolonged incubation beyond 60 min, labeled DNA molecules sedimenting more rapidly than mature, full-length virus genomes are observed. The integration of mutant DNA into mature virions is less rapid than that of the wide-type DNA. Upon extraction from the virosomes, the ts6389 DNA sediments as both genome-size and larger, faster sedimenting DNA. Upon treatment with restriction endonucleases, the ts6389 virosomal DNA exhibited an additional fragment after separation on agarose gels, perhaps as a consequence of fusion between the terminal fragments of the molecule. Taken together these observations suggest that concatemeric intermediates are formed during vaccinia DNA replication. By measuring the radioactivity incorporated into the fragments and subfragments of the molecules labeled during the first round of replication, the initiation site of replication can be localized to a region within the terminal 150 bp.     

Recombinant parainfluenza virus 5 (PIV5) expressing the influenza A virus hemagglutinin provides immunity in mice to influenza A virus challenge

Parainfluenza virus type 5 (PIV5), formerly known as simian virus 5 (SV5), is a non-segmented negative strand RNA virus that offers several advantages as a vaccine vector. PIV5 infects many cell types causing little cytopathic effect, it replicates in the cytoplasm of infected cells, and does not have a DNA phase in its life cycle thus avoiding the possibility of introducing foreign genes into the host DNA genome. Importantly, PIV5 can infect humans but it is not associated with any known human illness. PIV5 grows well in tissue culture cells, including Vero cells, which have been approved for vaccine production, and the virus can be obtained easily from the media. To test the feasibility of using PIV5 as a live vaccine vector, the hemagglutinin (HA) gene from influenza A virus strain A/Udorn/72 (H3N2) was inserted into the PIV5 genome as an extra gene between the hemagglutinin-neuraminidase (HN) gene and the large (L) polymerase gene. Recombinant PIV5 containing the HA gene of Udorn (rPIV5-H3) was recovered and it replicated similarly to wild type PIV5, both in vitro and in vivo. The HA protein expressed by rPIV5-H3-infected cells was incorporated into the virions and addition of the HA gene did not increase virus virulence in mice. The efficacy of rPIV5-H3 as a live vaccine was examined in 6-week-old BALB/c mice. The results show that a single dose inoculation provides broad and considerable immunity against influenza A virus infection.     

Wound healing in MIP-1alpha(-/-) and MCP-1(-/-) mice

A salient feature of normal wound healing is the development and resolution of an acute inflammatory response. Although much is known about the function of inflammatory cells within wounds, little is known about the chemotactic and activation signals that influence this response. As the CC chemokines macrophage inflammatory protein-1alpha (MIP-1alpha) and monocyte chemotactic protein-1 (MCP-1) are abundant in acute wounds, wound repair was examined in MIP-1alpha(-/-) and MCP-1(-/-) mice. Surprisingly, wound re-epithelialization, angiogenesis, and collagen synthesis in MIP-1alpha(-/-) mice was nearly identical to wild-type controls. In contrast, MCP-1(-/-) mice displayed significantly delayed wound re-epithelialization, with the greatest delay at day 3 after injury (28 +/- 5% versus 79 +/- 14% re-epithelialization, P < 0.005). Wound angiogenesis was also delayed in MCP-1(-/-) mice, with a 48% reduction in capillary density at day 5 after injury. Collagen synthesis was impeded as well, with the wounds of MCP-1(-/-) mice containing significantly less hydroxyproline than those of control mice (25 +/- 3 versus 50 +/- 8 microg/wound at day 5, P < 0.0001). No change in the number of wound macrophages was observed in MCP-1(-/-) mice, suggesting that monocyte recruitment into wounds is independent of this chemokine. The data suggest that MCP-1 plays a critical role in healing wounds, most likely by influencing the effector state of macrophages and other cell types.