Protective immunity against herpes simplex virus generated by DNA vaccination compared to natural infection.

To evaluate the utility of plasmid DNA vaccination against disease caused by herpes simplex virus (HSV), we compared the strength of protection against lethal challenge following natural virus infection with that following vaccination with a plasmid encoding HSV glycoprotein gD (gD-DNA). We further determined the cellular basis of each type of protection using lymphocyte deficient knockout mice. Establishment of immunity to HSV using live virus immunization required CD8+ T cells and B cells, but not CD4+ or gamma/delta+ T cells, and was related to specific antibody levels; surprisingly, CD4 knockout mice had large quantities of IgG anti-HSV serum antibodies. Establishment of immunity to HSV using gD-DNA immunization approached the strength of that generated following sublethal infection, but was dependent on alpha/beta+ CD4+ T cells, CD8+ T cells, B cells, and even partially on gamma/delta+ T cells, and not strictly correlated with antibody levels.

Susceptibility to secondary Francisella tularensis live vaccine strain infection in B-cell-deficient mice is associated with neutrophilia but not with defects in specific T-cell-mediated immunity.

Previous studies have demonstrated a role for B cells, not associated with antibody production, in protection against lethal secondary infection of mice with Francisella tularensis live vaccine strain (LVS). However, the mechanism by which B cells contribute to this protection is not known. To study the specific role of B cells during secondary LVS infection, we developed an in vitro culture system that mimics many of the same characteristics of in vivo infection. Using this culture system, we showed that B cells do not directly control LVS infection but that control of LVS growth is mediated primarily by LVS-primed T cells. Importantly, B cells were not required for the generation of effective memory T cells since LVS-primed, B-cell-deficient (BKO) mice generated CD4(+) and CD8(+) T cells that controlled LVS infection similarly to LVS-primed CD4(+) and CD8(+) T cells from wild-type mice. The control of LVS growth appeared to depend primarily on gamma interferon and nitric oxide and was similar in wild-type and BKO mice. Rather, the inability of BKO mice to survive secondary LVS infection was associated with marked neutrophil influx into the spleen very early after challenge. The neutrophilia was directly associated with B cells, since BKO mice reconstituted with naive B cells prior to a secondary challenge with LVS had decreased bacterial loads and neutrophils in the spleen and survived.

Protective immunity to Bordetella pertussis requires both B cells and CD4(+) T cells for key functions other than specific antibody production.

To investigate the fundamental nature of protective immunity to Bordetella pertussis, we studied intranasal immunization of adult mice with formalin-fixed B. pertussis (FFBP), followed by aerosol B. pertussis challenge. Mice given two doses of FFBP intranasally completely cleared a subsequent pertussis aerosol challenge from tracheae and lungs (defined as protection), but there was no correlation between levels of specific antibody and clearance of bacteria. Further, transfer of immune serum before aerosol challenge had minimal effects on bacterial burdens. However, pertussis-specific T cells producing interferon gamma but not interleukin 4 or interleukin 10 were detected in draining lymph nodes of FFBP-immunized mice. Significantly, repeated immunization of B cell knockout (BKO) mice resulted in partial protection, and complete protection was reconstituted by transfer of pertussis-immune B cells; reconstituted BKO mice had little if any detectable antipertussis antibodies. Immunization of mice lacking all T cells or lacking CD4(+) T cells did not lead to protection; in contrast, CD8(-) mice were protected. Mice depleted of CD4(+) T cells after immunization but before aerosol challenge, which thus had normal amounts of specific antibodies, were not optimally protected. Taken together, these data indicate that protective immunity to pertussis is dependent on both CD4(+) T cells and B cells, and both cell types provide significant functions other than specific antibody production.

Infection of B cell-deficient mice with CDC 1551, a clinical isolate of Mycobacterium tuberculosis: delay in dissemination and development of lung pathology.

Long-term survival of mice infected with Mycobacterium tuberculosis is dependent upon IFN-gamma and T cells, but events in early phases of the immune response are not well understood. In this study, we describe a role for B cells during early immune responses to infection with a clinical isolate of M. tuberculosis (CDC 1551). Following a low-dose infection with M. tuberculosis CDC 1551, similar numbers of bacteria were detected in the lungs of both B cell knockout (IgH 6-, BKO) and C57BL/6J (wild-type) mice. However, despite comparable bacterial loads in the lungs, less severe pulmonary granuloma formation and delayed dissemination of bacteria from lungs to peripheral organs were observed in BKO mice. BKO mice reconstituted with naive B cells, but not those given M. tuberculosis-specific Abs, before infection developed pulmonary granulomas and dissemination patterns similar to wild-type animals. Further analysis of lung cell populations revealed greater numbers of lymphocytes, especially CD8+ T cells, macrophages, and neutrophils in wild-type and reconstituted mice than in BKO mice. Thus, less severe lesion formation and delayed dissemination of bacteria found in BKO mice were dependent on B cells, not Abs, and were associated with altered cellular infiltrate to the lungs. These observations demonstrate an important, previously unappreciated, role for B cells during early immune responses to M. tuberculosis infections.

Detection of Francisella tularensis in infected mammals and vectors using a probe-based polymerase chain reaction.

We investigated the use of a TaqMan 5' nuclease assay (5NA) directed against the Francisella tularensis outer membrane protein (Fop) gene and a polymerase chain reaction-enzyme immunoassay (PCR-EIA) directed against the tul 4 gene for detection of this organism in experimentally infected mice and in field-collected tick vectors. We also evaluated the use of specially formulated filter paper (FTA) for rapid sample preparation. The 5NA had a detection limit of 1 pg of genomic DNA (<100 colony-forming units) and could be completed within several hours. The PCR-EIA could detect 1 pg of genomic DNA and 10 attograms (ag) (22 copies) of cloned insert, but takes longer to perform. Both assays were genus-specific, and successfully detected F. tularensis in mouse tissues (5NA) and in tick extracts (PCR-EIA). The FTA paper provided inexpensive, rapid, template preparation for the tick extracts, mouse tissues, and DNA obtained from clinical specimens. These probe-based assays have the potential to provide rapid, real-time/high-throughput molecular diagnostics in field situations.

Purified lipopolysaccharide from Francisella tularensis live vaccine strain (LVS) induces protective immunity against LVS infection that requires B cells and gamma interferon.

Previous results have demonstrated that nonspecific protective immunity against lethal Francisella tularensis live vaccine strain (LVS) or Listeria monocytogenes infection can be stimulated either by sublethal infection with bacteria or by treatment with bacterial DNA given 3 days before lethal challenge. Here we characterize the ability of purified lipopolysaccharide (LPS) from F. tularensis LVS to stimulate similar early protective immunity. Treatment of mice with surprisingly small amounts of LVS LPS resulted in very strong and long-lived protection against lethal LVS challenge within 2 to 3 days. Despite this strong protective response, LPS purified from F. tularensis LVS did not activate murine B cells for proliferation or polyclonal immunoglobulin secretion, nor did it activate murine splenocytes for secretion of interleukin-4 (IL-4), IL-6, IL-12, or gamma interferon (IFN-gamma). Immunization of mice with purified LVS LPS induced a weak specific anti-LPS immunoglobulin M (IgM) response and very little IgG; however, infection of mice with LVS bacteria resulted in vigorous IgM and IgG, particularly IgG2a, anti-LPS antibody responses. Studies using various immunodeficient mouse strains, including LPS-hyporesponsive C3H/HeJ mice, muMT(-) (B-cell-deficient) knockout mice, and IFN-gamma-deficient mice, demonstrated that the mechanism of protection does not involve recognition through the Lps(n) gene product; nonetheless, protection was dependent on B cells as well as IFN-gamma.

Importance of B cells, but not specific antibodies, in primary and secondary protective immunity to the intracellular bacterium Francisella tularensis live vaccine strain.

Although there appears to be little if any role for specific antibodies in protection against intracellular bacteria, such as the model pathogen F. tularensis live vaccine strain (LVS), the role of B cells themselves in primary and secondary infection with such bacteria has not been examined directly. We show here that mice deficient in mature B cells and antibodies (B-cell knockout mice) are marginally compromised in controlling primary sublethal infection but are 100-fold less well protected against secondary lethal challenge than are their normal counterparts. This defect in optimal specific protective immunity was readily reconstituted by the transfer of primed, and to a lesser degree, unprimed B cells, but not by the transfer of specific antibodies. The results indicate a previously unappreciated role for B cells in secondary immunity to intracellular pathogens through a function other than antibody production.

Bacterial DNA containing CpG motifs stimulates lymphocyte-dependent protection of mice against lethal infection with intracellular bacteria.

Bacterial DNA containing unmethylated CpG motifs activates mammalian lymphocytes and macrophages to produce cytokines and polyclonal Ig. These include IFN-gamma, IL-12, TNF-alpha, and IL-6, which are important in the control of intracellular bacterial infection. Here, we show that bacterial DNA, as well as synthetic oligonucleotides containing CpG motifs, induce protection against large lethal doses of Francisella tularensis live vaccine strain (LVS) and Listeria monocytogenes. Methylation of DNA at CpG dinucleotides or inversion of the motif abolished this protection. Surprisingly, DNA-mediated protection was highly dependent on lymphocytes, particularly B cells, as well as the production of IFN-gamma. Optimal protection was elicited 2-3 days after inoculation with DNA and persisted for up to 2 wk. Further, animals surviving lethal challenge developed pathogen-specific secondary immunity. These findings indicate that host innate immune responses to bacterial DNA may contribute to the induction of protective immunity to bacteria and the subsequent development of memory.

Antibody response and protective capacity of plasmid vaccines expressing three different herpes simplex virus glycoproteins.

Plasmid expression vectors were constructed that contained the genes encoding herpes simplex virus 1 (HSV-1) glycoproteins C (gC), D (gD), and E (gE). Mice receiving two intramuscular injections of expression plasmid (50 microg) produced a specific HSV-1 antibody response. Mice receiving the gD plasmid were protected against a lethal intraperitoneal challenge of HSV-1 (5 x 10(4) pfu) but not against more demanding challenge doses. Protection with gC or gE plasmid vaccination could be demonstrated only if the inoculating dose of DNA was increased to 250 microg. In contrast, all mice immunized with vaccinia recombinants expressing either gC or gE survived HSV-1 challenge. Analysis of the HSV-1 antibody isotype produced by plasmid immunization revealed a response dominated by IgG2a. Combination delivery of all three glycoprotein expression plasmids provided better protection against lethal challenge, but mice receiving the combination were still not able to withstand increased challenge doses of virus.

Nonspecific early protective immunity in Francisella and Listeria infections can be dependent on lymphocytes.

Normal mice, but not lymphocyte-deficient or B-cell-deficient mice, given a sublethal infection of Francisella tularensis LVS survive a secondary lethal challenge of more than 10,000 50% lethal doses given 3 days later. In this work, we show that similar early protection that is also strongly lymphocyte dependent operates in Listeria monocytogenes infection. Since sublethal infection with either LVS or L. monocytogenes protects against heterologous lethal challenge, this early protection is nonspecific.

A novel role for B cells in early protective immunity to an intracellular pathogen, Francisella tularensis strain LVS.

Normal BALB/cByJ mice given a sublethal infection of Francisella tularensis strain LVS survived 10(6) LD50s of lethal challenge given only 3 days later. Here, we determine the cell types responsible for this very strong early protective immunity. Early protection is observed in athymic nu/nu mice but not fully immunodeficient scid mice, implicating a lymphocyte in this response. Using scid mice that are reconstituted with various purified cell subpopulations, as well as mice with genetically targeted disruptions in lymphocyte subpopulations (knockout mice), we demonstrate that strong early protection is highly dependent on B cells. This protective mechanism, which limits bacterial growth in the organs of the reticuloendothelial system very quickly after infection, requires IFN-gamma but is unlikely to involve specific Ab.

Loss of either CD4+ or CD8+ T cells does not affect the magnitude of protective immunity to an intracellular pathogen, Francisella tularensis strain LVS.

Normal mice readily survive a sublethal intradermal (i.d.) infection with Francisella tularensis live vaccine strain (LVS), a model intracellular bacterium, and are strongly protected against subsequent lethal challenge. However, athymic nu/nu mice, which lack mature alphabeta TCR+ T lymphocytes, succumb to i.d. infection within 30 days. Here we characterize the alphabeta T cell subpopulations necessary for both resolution of i.d. infection and generation of optimal protective immunity to LVS. BALB/cByJ mice treated with anti-CD4 or anti-CD8 Abs before i.d. infection survived and cleared bacteria, and anti-CD4- or anti-CD8-treated immune mice survived a very strong i.p. challenge of 10,000 LD50s. Among mutant mice with targeted gene disruptions (knockouts), CD4-, beta2-microglobulin-deficient (which are also CD8-), and gammadelta TCR- mice all resolved a large sublethal i.d. infection. All CD4- and beta2-microglobulin-deficient mice readily survived subsequent lethal i.p. challenge of 10,000 LD50s, even in the absence of specific IgG Abs, as did most (86%) gammadelta TCR- mice. In contrast, alphabeta TCR- mice or alphabeta + gammadelta TCR- mice died about 35 days after i.d. infection. Depletion of gammadelta+ T cells from alphabeta TCR- mice had no effect on mean time to death from i.d. LVS infection. Therefore alphabeta TCR+ cells are required for protection, but either CD4+ or CD8+ T cells are individually sufficient to resolve a large sublethal i.d. LVS infection and to protect against a maximal secondary lethal challenge. These results emphasize the remarkable plasticity of the alphabeta T cell response in protective immunity to intracellular bacteria.

Minimal requirements for murine resistance to infection with Francisella tularensis LVS.

Intraperitoneal or intravenous infection of mice with Francisella tularensis LVS is lethal, with an intraperitoneal 50% lethal dose (LD50) approaching a single bacterium. Intradermal (i.d.) LVS infection has a much higher LD50, about 10(6) bacteria in BALB/cByJ mice, and survival of i.d. infection leads to solid generation of immunity against lethal challenge. To define the minimal requirements for both initial and long-term survival of i.d. infection, we characterized the nature of i.d. LVS infection in lymphocyte-deficient BALB/cByJ.scid (scid) mice. scid mice infected i.d. with strain LVS survived for about 20 days and then died from overwhelming disseminated infection. However, scid mice treated with monoclonal antibodies to gamma interferon, tumor necrosis factor alpha, or neutrophils-granulocytes all died within 1 week of infection, indicating that these were essential for early control of infection. Studies using GKO (gamma interferon knockout) mice emphasized that gamma interferon is absolutely required for initial survival of i.d. LVS infection. scid mice could be reconstituted for long-term survival of i.d. LVS infection and clearance of bacteria by intravenous transfer of splenic lymphocytes or purified B220-/T+ lymphocytes but not nu/nu lymphocytes. T cells are therefore required for long-term clearance and survival of i.d. LVS infection; efforts to determine whether CD4+ T cells, CD8+ T cells, or both are involved are ongoing.

Effects of exogenous, nonleukemogenic, ecotropic murine leukemia virus infections on the immune systems of adult C57BL/6 mice.

Mouse AIDS (MAIDS) develops in mice infected with a mixture of replication-competent ecotropic and mink lung cell focus-inducing murine leukemia viruses and an etiologic replication-defective virus. Helper viruses are not required for induction of MAIDS, but the time course of disease is accelerated in their presence. To understand the possible contributions of ectropic murine leukemia viruses to MAIDS pathogenesis, we biologically cloned a series of viruses from the MAIDS-inducing LP-BM5 virus mixture. These viruses were examined for replication in tissues of infected mice and for effects on the immune system. All virus stocks replicated efficiently in mice. Infected animals showed slight lymphadenopathy and splenomegaly due primarily to B-cell proliferation associated with differentiation to immunoglobulin secretion resulting in twofold increases in serum immunoglobulin M levels; however, B-cell responses to helper T-cell-independent antigens were increased rather than decreased as in MAIDS. Analyses of CD8+ T-cell function showed that cytotoxic T-lymphocyte responses to alloantigens were comparable in control and infected mice. Finally, we showed that infection resulted in enhanced expression of transcripts for interleukin-10, interleukin-4, and gamma interferon. These cytokines can all contribute to B-cell activation and may promote the expansion of a target cell population for the MAIDS defective virus.

Heat stress alters the virulence of a rifampin-resistant mutant of Francisella tularensis LVS.

We have studied the stress response of a rifampin-resistant mutant of Francisella tularensis LVS. This mutant, Rif 7, was avirulent with an intraperitoneally administered 50% lethal dose greater than 10(7) CFU in a murine model of infection. Exposure of Rif 7 to heat stress for 5 h in vitro resulted in a 2-log decrease in its 50% lethal dose (P < 0.02). The increase in virulence was dependent on the time of exposure to high temperature and was maximal at 5 h. Envelope preparations from heat-stressed cells showed increased levels of several proteins. Notable among these were polypeptides with approximate molecular masses of 16, 60, and 75 kDa. Increases in both virulence and envelope protein levels were reversed when heat-treated cells were subsequently grown at 37 degrees C. Inhibition of protein synthesis by actinomycin D during heat stress blocked the increase in virulence of Rif 7. Cell-free media from the heat-stressed Rif 7 reacted with the whole spectrum of bacterial proteins were not toxic to mice. Hyperimmune serum against Rif 7 reacted with the whole spectrum of bacterial proteins in Western blots (immunoblots), although its reaction with 34- and 45-kDa proteins and two 60- and 75-kDa proteins upregulated during heat stress was weak. Other stress conditions, low iron and low pH, caused similar increases in the virulence of Rif 7. However, examination of the protein profile did not reveal any major common polypeptides induced by different stresses. Heat-treated Rif 7 bacteria were fully able to replicate in macrophages in vitro and in the host tissues, even though heat treatment only partially restored virulence.

Transfer of immunity against lethal murine Francisella infection by specific antibody depends on host gamma interferon and T cells.

Both serum and spleen cells from mice immune to Francisella tularensis transfer protection to naive recipients. Here we characterize the mechanism of protection induced by transfer of immune mouse serum (IMS). IMS obtained 4 weeks after intradermal infection with 10(3) bacteria of the live vaccine strain (LVS) contained high levels of immunoglobulin G2 (IgG2a) and IgM (end point titers, 1:16,600 and 1:7,200, respectively) and little IgG1, IgG2b, or IgG3. LVS-specific antibodies were detected 5 days after intradermal infection, and reached peak levels by 2 weeks postinfection. Only sera obtained 10 days or more after sublethal infection, when IgG titers peaked, transferred protection against a challenge of 100 50% lethal doses (LD50s). Purified high-titer IgG anti-LVS antibody but not IgM anti-LVS antibody was responsible for transfer of protection against an intraperitoneal challenge of up to 3,000 LD50s. IMS had no direct toxic effects on LVS and did not affect uptake or growth of bacteria in association with peritoneal cells. One day after LVS infection, liver, spleen, and lung tissue from mice treated with IMS contained 1 to 2 log units fewer bacteria than did tissue from mice treated with normal mouse serum or phosphate-buffered saline. Between 2 and 4 days after infection, however, bacterial growth rates in tissues were similar in both serum-protected mice and unprotected mice. Bacterial burdens in IMS-treated, LVS-infected mice declined in infected tissues after day 5, whereas control animals died. This lag phase suggested that development of a host response was involved in complete bacterial clearance. In fact, transfer of IMS into normal recipients that were simultaneously treated with anti-gamma interferon and challenged with LVS did not protect mice from death. Further, transfer of IMS into athymic nu/nu mice did not protect against LVS challenge; protection was, however, reconstituted by transfer of normal T cells into nu/nu mice. Thus, "passive" transfer of protection against LVS with specific antibody is not passive but depends on a host T-cell response to promote clearance of systemic infection and protection against lethal disease.

In vivo delivery of interleukin-4 by a recombinant vaccinia virus prevents tumor development in mice.

To study the immunotherapeutic potential of interleukin-4 (IL-4) delivered in vivo via a recombinant vaccinia virus, a thymidine kinase-negative (TK-) vaccinia virus that expressed the murine IL-4 gene (VV1/IL-4) was constructed. When mice were inoculated with 10(7) plaque-forming units (pfu) of VV1/IL-4 subcutaneously (s.c.), 10(5) pfu/cm2 were found in skin, and smaller numbers in liver and kidney between 1 and 7 days after infection; few viral pfu were found in spleen and lung, or in any organ after intravenous infection. This suggested that recombinant vaccinia viruses might be most efficient at delivery of cytokine genes to the skin. Because IL-4 has recently been found to have potent anti-tumor activity, the effect of recombinant virus infection on the development of s.c. tumors was studied. A single s.c. inoculation with VV1/IL-4 delayed the development of NCTC 2472 tumors, but when VV1/IL-4 was inoculated s.c. weekly for 8 weeks, tumor development was completely prevented in 93% of mice. Similarly, the development of M-3 melanoma tumors was also prevented by weekly s.c. inoculations of VV1/IL-4. About 40% of mice treated with control VV2/beta gal by the same regimen also failed to develop tumors. Weekly virus treatment did not prevent NCTC 2472 tumor development in athymic nu/nu mice, suggesting that mature T cells are required for expression of VV1/IL-4 induced antitumor activity. Thus, recombinant vaccinia viruses may be especially well suited for convenient therapeutic delivery of immunomodulator genes to skin-related sites.

Replication-incompetent herpesvirus vector delivery of an interferon alpha gene inhibits human immunodeficiency virus replication in human monocytes.

Human monocytes and macrophages are nondividing cells that serve as a major reservoir for human immunodeficiency virus (HIV) at all stages of infection. To investigate viral-mediated gene delivery as a means of inhibiting HIV replication in human monocytes, a replication-incompetent herpes simplex virus vector was developed that expressed human interferon alpha. Monocytes infected with this herpes simplex virus vector and then challenged with HIV showed dramatically reduced cytopathic effects and HIV replication compared to control treated monocytes. Similar effects on HIV replication were observed if monocytes were first infected with HIV and then treated with the recombinant vectors. These results demonstrate that replication-incompetent herpes simplex virus gene delivery of interferon alpha directly to human monocytes can greatly decrease HIV replication and suggest that such a vector might deliver therapeutically important genes directly to sites of HIV infection.

T-cell-independent resistance to infection and generation of immunity to Francisella tularensis.

The intraperitoneal 50% lethal dose (LD50) for Francisella tularensis LVS in both normal control heterozygote BALB/c nu/+ mice and BALB/c nu/nu mice was 2 x 10(0). Both nu/+ and nu/nu mice given 10(7) LVS bacteria or more intradermally (i.d.) died, with a mean time to death of about 7 to 8 days. On the other hand, nu/+ mice given 10(6) LVS bacteria or less survived for more than 60 days and cleared systemic bacteria, while nu/nu mice given 10(6) LVS bacteria or less survived for more than 10 days but died between days 25 and 30. Thus, the short-term (i.e., < 10-day) i.d. LD50 of both nu/nu and nu/+ mice was 3 x 10(6), but the long-term (i.e., > 10-day) i.d. LD50 of nu/nu mice was less than 7 x 10(0). The short-term survival of i.d. infection was dependent on tumor necrosis factor and gamma interferon: treatment of nu/nu mice with anti-tumor necrosis factor or anti-gamma interferon at the time of i.d. infection resulted in death from infection 7 to 8 days later, whereas control infected nu/nu mice survived for 26 days. nu/nu mice infected with LVS i.d. generated LVS-specific serum antibodies, which were predominantly immunoglobulin M: titers peaked 7 days after i.d. infection but declined sharply by day 21, after which mice died. Surprisingly, nu/nu mice given 10(3) LVS bacteria i.d. became resistant to a lethal challenge (5,000 LD50s) of LVS intraperitoneally within 2 days after i.d. infection; nu/nu mice similarly infected with LVS i.d. and challenged with Salmonella typhimurium (10 LD50s) were not protected. nu/nu mice given nu/+ spleen cells intravenously as a source of mature T cells survived i.d. infection for more than 60 days and cleared bacteria. Taken together, these studies demonstrate that i.d. infection of nu/nu mice with LVS rapidly generates T-cell-independent, short-term, specific protective immunity against lethal challenge, but T lymphocytes are essential for long-term survival.