Immunity in the female genital tract after intravaginal vaccination of mice with an attenuated strain of herpes simplex virus type 2.

Herpes simplex virus type 2 is a common human venereal pathogen which causes lethal neurological illness after intravaginal inoculation into BALB/cJ mice. To investigate whether an attenuated, nonlethal strain of this virus would confer immunity after inoculation of mice, we constructed a strain containing a partial deletion of the thymidine kinase gene, which is necessary for viral replication and spread in sensory ganglia. Unlike its wild-type counterpart, this deletion-containing strain of herpes simplex virus type 2 caused mild clinical disease and was not lethal when studied in an age-dependent murine model of intravaginal infection. Furthermore, after intravaginal infection, the deletion-containing strain could not be isolated from sensory ganglia at a time when wild-type virus was abundant. Of greater significance, intravaginal inoculation with the deletion-containing strain rendered mice completely resistant to rechallenge with a 10-fold 50% lethal dose of wild-type virus. These results suggest that a strain of herpes simplex virus type 2 containing a deletion of the thymidine kinase gene will be useful in studying the cellular basis of mucosal immunity in the genital tract.     

Protective effect of measles virus inoculation on subacute sclerosing panencephalitis virus-infected mice

The Biken strain of subacute sclerosing panencephalitis (SSPE) virus caused a fatal neurologic disease in adult mice after intracerebral inoculation. However, the mice were completely protected from the disease when a high dose of measles virus was given intracerebrally after the SSPE virus infection. The measles virus inoculation induced interferon production and immune responses. An experiment with athymic nude mice showed that interferon and anti-measles antibody were able to prolong the incubation period of the disease but not to protect the SSPE virus-infected nude mice from death. For complete protection, T lymphocytes appeared to be essential. The present study suggested that the protective effect of measles virus inoculation is basically due to the induction of immune responses and that SSPE virus infection in mice is susceptible to immune reactions.     

Functional analysis of T lymphocyte subsets in antiviral host defense

The role of different T cell subsets in antiviral host defense was investigated by treating thymectomized C57BL/6 and CBA/J mice with monoclonal rat anti-Lyt-2 or anti-L3/T4 IgG 2b antibodies 14 and 10 days before infection. This treatment depleted the respective T cell subsets to undetectable levels in peripheral blood when assayed by immunofluorescence. In mice treated with anti-Lyt-2, induction of cytotoxic T cells was reduced to less than 1 to 2% after intravenous infection with Armstrong strain of lymphocytic choriomeningitis virus (LCMV). In addition, no primary swelling of the footpad could be detected following local inoculation of the virus. In animals treated with anti-L3/T4, antiviral cytotoxic T lymphocyte responses were reduced by a factor of 10. These L3/T4+ cell-depleted mice showed delayed footpad swelling after local injection of LCMV Armstrong. After intracerebral infection with LCMV, anti-Lyt-2-treated mice were resistant and those injected with anti-L3/T4 were totally susceptible to LCMV Armstrong-triggered immunopathologic disease. Virus could be detected in the blood of antibody-treated mice 7 days after inoculation; however, no virus could be measured in the blood of surviving anti-Lyt-2-treated animals 15 days after intracerebral infection. Serum titers of interferon-alpha,beta induced by viral infection remained unaffected by depletion of T cell subsets. Anti-L3/T4 antibody-treated C57BL/6 mice failed to generate IgG antibodies against the New Jersey strain of vesicular stomatitis virus, whereas Lyt-2+ cell-depleted mice had normal antivesicular stomatitis virus (New Jersey strain) IgG antibody titers.     

Role of herpes simplex virus 1 Us3 in viral neuroinvasiveness

Us3 is a serine–threonine protein kinase that is encoded by herpes simplex virus 1 (HSV‐1). In experimental animal models of HSV infection, peripheral and intracranial inoculations can be used to study viral pathogenicity in peripheral sites (e.g., eyes and vagina) and central nervous systems (CNSs), respectively. In addition, peripheral inoculation can be used to investigate this virus' ability to invade the CNS (neuroinvasiveness) from peripheral sites. HSV‐1 Us3 has previously been shown to be critical for viral pathogenicity in both peripheral sites and CNSs of mice. However, the role of HSV‐1 Us3 in viral neuroinvasiveness has not yet been elucidated. In the present study, the yields of a Us3 null mutant virus and its repaired virus in the eyes, trigeminal ganglia, and brains of mice following ocular inoculation were examined. It was found that, although the repaired virus appeared in the brains of mice 3 days after infection, peak replication occurring 7 days after infection, no viral replication of the Us3 null mutant virus was detectable. These findings indicate that HSV‐1 Us3 plays a crucial role in the ability of the virus to invade the brain from the eyes. Thus, HSV‐1 Us3 is a significant neuroinvasiveness factor in vivo.     

Attenuated, replication-competent herpes simplex virus type 1 mutant G207: safety evaluation in mice

Herpes simplex virus type 1 (HSV-1) mutants that are attenuated for neurovirulence are being used for the treatment of cancer. We have examined the safety of G207, a multimutated replication-competent HSV-1 vector, in mice. BALB/c mice inoculated intracerebrally or intracerebroventricularly with 10(7) PFU of G207 survived for over 20 weeks with no apparent symptoms of disease. In contrast, over 80% of animals inoculated intracerebrally with 1.5 x 10(3) PFU of HSV-1 wild-type strain KOS and 50% of animals inoculated intracerebroventricularly with 10(4) PFU of wild-type strain F died within 10 days. Similarly, after intrahepatic inoculation of G207 (3 x 10(7) PFU) all animals survived for over 10 weeks, whereas no animals survived for even 1 week after inoculation with 10(6) PFU of KOS. After intracerebroventricular inoculation, LacZ expression was initially observed in the cells lining the ventricles and subarachnoid space; expression decreased until almost absent within 5 days postinfection, with no apparent loss of ependymal cells. G207 DNA could be detected by PCR in the brains of mice 8 weeks after intracerebral inoculation; however, no infectious virus could be detected after 2 days. As a model for latent HSV in the brain, we used survivors of an intracerebral inoculation of HSV-1 KOS at the 50% lethal dose. Inoculation of a high dose of G207 at the same stereotactic coordinates did not result in reactivation of detectable infectious virus or symptoms of disease. We conclude that G207 is safe at or above doses that were efficacious in mouse tumor studies.     

Infection with Cytotoxic T-Lymphocyte Escape Mutants Results in Increased Mortality and Growth Retardation in Mice Infected with a Neurotropic Coronavirus

C57BL/6 mice infected with mouse hepatitis virus strain JHM (MHV-JHM) develop a chronic demyelinating encephalomyelitis several weeks after inoculation. Previously, we showed that mutations in the immunodominant CD8 T-cell epitope (S-510-518) could be detected in nearly all samples of RNA and virus isolated from these mice. These mutations abrogated recognition by T cells harvested from the central nervous systems of infected mice in direct ex vivo cytotoxicity assays. These results suggested that cytotoxic T-lymphocyte (CTL) escape mutants contributed to virus amplification and the development of clinical disease in mice infected with wild-type virus. In the present study, the importance of these mutations was further evaluated by infecting naive mice with MHV-JHM variants isolated from infected mice and in which epitope S-510-518 was mutated. Compared to mice infected with wild-type virus, variant virus-infected animals showed higher mortality and morbidity manifested by decreased weight gain and neurological signs. Although a delay in the kinetics of virus clearance has been demonstrated in previous studies of CTL escape mutants, this is the first illustration of significant changes in clinical disease resulting from infection with viruses able to evade the CD8 T-cell immune response.     

Role of mink cell focus-inducing virus in leukemias induced by Friend ecotropic virus

Recombinant viruses have been implicated in the pathogenesis of murine leukemias induced by a variety of long-latency retroviruses. Neonatal mice of several strains were inoculated with Friend ecotropic virus (F-Eco) and analyzed for the presence of mink cell focus-inducing (MCF) virus or DNA restriction enzyme fragments which were specific for Friend MCF virus (F-MCF). MCF virus was detected within 2 weeks of inoculation in NFS /N mice and at about 2 months after inoculation in BALB/c mice. Both of these strains developed erythroblastosis after inoculation with F-Eco. In contrast, MCF virus was not detected in F-Eco-inoculated C57BL mice. These mice were resistant to erythroblastosis but developed lymphoma or myelogenous leukemia or both at about 5 months after inoculation. Thus, although MCF viruses were associated with F-Eco erythroblastosis in NFS /N and BALB/c mice, they were not necessary for F-Eco-induced lymphoid or myeloid leukemias in C57BL mice. To investigate the association between resistance to erythroblastosis and absence of MCF virus, C57BL mice were inoculated with pseudotypic mixtures of F-Eco plus F-MCF; MCF virus replicated well in these mice, but the mice remained resistant to erythroblastosis. Furthermore, in genetic crosses between C57BL and NFS /N or BALB/c, some mice inherited resistance to F-Eco erythroblastosis without inheriting the C57BL resistance to the generation of MCF viruses. These results indicate that C57BL mice carry a gene for resistance to F-Eco erythroblastosis which is distinct from the C57BL genes which interfere with the generation of MCF viruses.     

An Adenovirus Isolated from the Feces of Mice

Experimental infection of an inbred strain of DK1 mice was carried out with a mouse adenovirus strain K87, isolated from the feces of apparently healthy DK1 mice. Strain K87 was orally administered to four-week-old mice and the virus was recovered from their feces for at least 3 weeks. The highest virus titers in the feces were observed between 1 and 2 weeks after inoculation. In 7-week-old mice the period of virus excretion was about one week shorter. When neonatal or 2-week-old mice were administered virus orally, somewhat irregular results but similar to those from the 4 or 7-week-old mice were obtained. In these infected mice, virus growth was detected in the intestinal tract but not in oropharyngeal washings, nasal tissue, lung, spleen or urine. After inoculation through several parenteral routes, the virus was also detected in the feces and virus growth seemed to be limited mainly to the intestinal tract. No clinical manifestations were observed in any infected mice. When the virus was almost undetectable in the mice infected either orally or parenterally, the virus was readministered orally, but no further virus was recovered in the feces. Neutralizing antibody in the serum was detected 3 weeks after primary inoculation. Induction of immunological tolerance was examined by inoculating mice in utero 2–4 days before birth, but no evidence of induced tolerance was obtained. The use of the adenovirus strain K87-mouse system as a model system for the study of the infectious process and immune mechanisms is suggested because strain K87 has a tissue tropism analogous to many human adenoviruses.     

Pathogenesis of street rabies virus infections in resistant and susceptible strains of mice.

Seven strains of mice were examined to determine why susceptibility differences and variations in clinical central nervous system (CNS) disease occurred among these animals after intraperitoneal inoculation of street rabies virus (SRV). Trace experiments for infectious virus indicated that these differences were associated with restriction of virus replication within the CNS. Limitation of viral replication appeared to correlate with the antibody response in that prominent serum anti-SRV neutralizing antibody titers were detected in resistant strains, whereas susceptible strains produced minimal amounts of antibody until their death. The importance of the immune response was reaffirmed with cyclophosphamide studies in that all resistant SJL/J mice died after immunosuppressive treatment. In contrast, cyclophosphamide-treated SJL/J mice whose immune systems were reconstituted with either unfractionated immune spleen cells or with sera 24 h after SRV inoculation survived a lethal dose of SRV. More importantly, immunosuppressed SJL/J and immunodeficient athymic mice were protected when reconstituted with immune serum 72 h after SRV inoculation, a time in which infectious virus was detected in the spinal cords of some mice but was not present in the peritoneal cavity. Additional studies showed that antibody in the cerebrospinal fluid was unimportant in the resistance of mouse strains which remained clinically asymptomatic, but it appeared to be associated with the survival of mice which developed clinical CNS disease. Furthermore, CNS resistance to intranasal or intracerebral inoculation with challenge virus standard rabies virus developed as early as 5 days post-intraperitoneal inoculation of SRV.     

Herpes simplex virus type 1-induced hydrocephalus in mice.

Adult ICR/Slc or BALB/c mice developed hydrocephalus when attenuated herpes simplex virus type 1 (HSV-1) (strain Ska) was injected intracerebrally 2 to 4 weeks earlier and then after mice were challenged with the same virus or virulent HSV-1. Initial inoculation of the Ska strain elicited acute meningitis and ependymitis with transient mild hydrocephalus. Viral antigen was seen in the meninges and subependymal areas, and the virus was titrated during the acute phase of infection. After the second virus inoculation, more prominent inflammation was evoked in the same area, and the animals developed hydrocephalus, although viral antigen and infectious virus were hardly detected. When the mice were immunosuppressed with cyclophosphamide, they ceased to develop hydrocephalus. BALB/c nude mice did not show the same pathology, even though they were treated in the same way. When irradiated mice, which had been infected with the Ska strain intracerebrally 2 weeks earlier, received syngeneic immune spleen cells, they developed hydrocephalus. The T-cell nature of the effector cells was confirmed by the elimination of the pathology after treatment of the donor cells with anti-Thy-1.2 plus complement. No hydrocephalic mice were observed after treatment of the donor cells with anti-Lyt-1.2 plus complement, which gave further evidence of the T-cell nature of the effector cells as the Lyt-1+.2+ antigen-bearing subsets. Intervals between priming and challenge virus inoculation could be more than 18 months. The presence of purified HSV-1 envelope protein was feasible for the development of the hydrocephalic animals.     

Non-invasive imaging of mouse hepatitis coronavirus infection reveals determinants of viral replication and spread in vivo

Bioluminescence imaging (BLI) is a powerful new method to study virus dissemination in the live animal. Here we used this method to monitor the spatial and temporal progression of mouse hepatitis coronavirus (MHV) infection in mice using luciferase-expressing viruses. Upon intranasal inoculation, virus replication could initially be observed in the nasal cavity and the cervical lymph nodes, after which the infection spread to the brain and frequently to the eyes. The kinetics of virus spread to and clearance from the brain appeared to depend on the inoculation dose. After intraperitoneal inoculation, virus replication was predominantly observed in the liver and occasionally in the intestines, but interestingly also in the tail and paws. BLI thus elucidated new anatomic locations of virus replication. Furthermore, MHV dissemination was shown to be critically depended on the viral spike protein, but also on the mouse strain used. Widespread dissemination was observed in mice lacking a functional type I interferon response. The importance of the type I interferon system in limiting viral spread was also demonstrated by the administration of type I interferons to mice. Our results provide new insights in coronavirus pathogenesis and demonstrate the potential of BLI to study coronavirus–host interactions in vivo .     

Lactate dehydrogenase-elevating agent is responsible for interferon induction and enhancement of natural killer cell activity by inoculation of Ehrlich ascites carcinoma cells into mice

Inoculation of Ehrlich ascites carcinoma cells (EAC) into the peritoneal cavities of outbred ddY mice induced interferon (IFN) in the circulation. The maximum titer (1,280 U) was obtained at 24 hr after inoculation. This induced IFN had the characteristics of type I IFN, i.e., stability at pH2 and lability at 56 C. An increase in natural killer cell (NK) activity was also observed for the first 3 days after inoculation. In addition, plasma lactate dehydrogenase (LDH) activity was elevated in these mice. Inoculation of ascitic fluid or serum of EAC-bearing mice into normal mice increased plasma LDH activity six- to sevenfold over normal levels and elevated activities persisted throughout the life of the mice. These results suggest that the LDH-elevating agent was responsible for IFN induction and for enhancing NK activity. Because lactate dehydrogenase-elevating virus (LDV) can be eliminated from tumor cells by passage in vitro, we attempted to grow EAC in tissue culture for several months and re-examined whether the inoculation of such cells could elevate plasma LDH activity induce IFN and enhance NK activity. The results showed that inoculation of the passaged cells had no effect on these activities in normal mice. Therefore, we concluded that the IFN inducer was LDV which contaminated the EAC and then enhanced the NK activity. N-tropic murine leukemia virus also contaminated EAC, but this virus was not responsible because cultured cells of EAC still shed this virus.     

Exposure to 2500 Lux Increases Serum Melatonin in Venezuelan Equine Encephalomyelitis

When mice infected with the Venezuelan equine encephalomyelitis (VEE) virus were exposed to 2500 lux with a 12 h light: 12 h dark photoperiod, the serum levels of melatonin (MLT) remained constantly elevated. In mice exposed to 400 lux low levels of serum MLT were detected during the day and high levels during the night. An increase in the survival rate of the infected mice from 6 to 13 days after virus inoculation was also observed. The significant increment in the concentration of serum MLT produced by the high intensity light could be responsible for the longer survival rate of mice infected with the VEE virus.     

Immunocontraception is induced in BALB/c mice inoculated with murine cytomegalovirus expressing mouse zona pellucida 3

Immunocontraception, the prevention of oocyte fertilization through immunological means, could potentially be used to control plaguing mouse populations in Australia. This paper describes the construction of a mouse-specific betaherpesvirus, murine cytomegalovirus, which has been engineered to express the murine zona pellucida 3 (ZP3) gene. A single inoculation of this recombinant virus resulted in almost complete infertility, persistent anti-ZP3 antibody production, and profound changes to ovarian morphology in BALB/c mice in the absence of significant virus replication during the acute phase of infection. Murine cytomegalovirus may prove to be useful as a vector for the delivery of a mouse-specific immunocontraceptive agent to target populations of wild mice in the field.     

Impairment of splenic natural killer cell activity of mice infected with the polycythemic strain of friend leukemia virus

Summary The natural killer (NK) cell activity of DBA/2J mice infected with the polycythemic variant of Friend leukemia virus (FLV-P) declines early after virus challenge, being non-detectable 2 weeks after FLV-P inoculation. Cells capable of suppressing the in vitro cytolytic activity of NK effector lymphocytes were detected in the spleen of infected mice. The mechanism of suppression could be based on cell-mediated impairment of NK effector function, or on antigenic competition from splenocytes expressing NK antigens elicited by FLV-P.     

Dissociation between lymphoproliferative responses and virus replication in mice with different sensitivities to retrovirus-induced immunodeficiency.

Murine AIDS (MAIDS) is induced by a replication-defective virus (BM5d). In susceptible mice (C57BL/6J), inoculation with LP-BM5 murine leukemia virus, which consists of the BM5d virus and replication-competent B-tropic ecotropic (BM5e) and milk cell focus-inducing (BM5-MCF) helper viruses results in the polyclonal proliferation of T and B cells, immunodeficiency, and the expansion of B cells containing the BM5d provirus followed by the development of B-cell lymphomas. Several strains of mice that are resistant to LP-BM5-induced murine AIDS have been identified, and major histocompatibility complex genes as well as non-major histocompatibility complex genes were shown to play a role in this resistance. In the present study, we have examined and compared the replication of the BM5d and BM5e viruses after inoculation of LP-BM5 into sensitive (C57BL/6J) and resistant (C57BL/KSJ) mice. Using a specific polymerase chain reaction, we could detect the BM5d and BM5e proviruses as early as 1 week postinfection in the sensitive mice, and the levels of both viruses increased significantly with the progression of the disease. In contrast, in the resistant C57BL/KSJ mice, replication of BM5d and BM5e was restricted and no BM5d and only very low levels of the BM5e provirus could be detected either at early or late times postinoculation with the LP-BM5 virus mixture. Inoculation with LP-BM5 did not lead to the production of antibodies that could recognize the BM5d-encoded Pr60gag in either the sensitive or resistant mice; however, production of antibodies recognizing the env-related proteins of the helper virus was detected in the resistant but not in the sensitive mice at late times postinfection. Interestingly, inoculation with LP-BM5 increased polyclonal stimulation of spleen cells and decreased mitogen stimulation in both strains of mice. This stimulation of splenocytes persisted in the sensitive mice but decreased after a few weeks in the resistant mice. These results show an early block in BM5d and BM5e replication in the resistant C57BL/KSJ mice and indicate that resistance is a consequence of the inhibition of an onset of the BM5d virus infection and its expansion. However, initial responses to virus infection such as proliferation of spleen cells and response to mitogen are similar in both strains of mice and are therefore not necessarily related to the development of the disease.     

Spread of herpes simplex virus in lymph nodes after experimental infection of mice

Herpes simplex virus was frequently isolated from ipsilateral popliteal lymph nodes after percutaneous inoculation of the dorsal face of the footpad, and from ipsi- and contralateral submandibular lymph nodes after percutaneous inoculation of the cheek or the orofacial area of mice. Virus was detected only on very rare occasion in nondraining lymph nodes (inguinal or axillary) or in contralateral popliteal lymph nodes, but was frequently isolated in contralateral lumbar lymph nodes after footpad inoculation. The presence of virus in lymph nodes paralleled or followed the invasion of ipsilateral sensory ganglia and was associated with dissemination of virus in contralateral sensory ganglia after unilateral inoculation. In older mice virus was detected only occasionally in lymph nodes and dissemination of virus in contralateral sensory ganglia was generally not observed. The results suggest that lymphatic spread may contribute to dissemination of virus in contralateral sensory ganglia after unilateral inoculation of mice.     

Antiviral activity of a thymic factor in experimental viral infections. I. Thymic hormonal effect on survival, interferon production and NK cell activity in Mengo virus-infected mice

A partially purified thymic factor, thymostimulin (TS), significantly increased the survival rate of adult, immune-intact mice infected with the neurotropic Mengo virus. TS treatment was begun after virus inoculation by daily i.p. injections. In untreated C57BL/6 mice, LD50 was reached with 1 X 10(4) PFU, but 10-fold more virus (i.e., 1 X 10(5) PFU) was needed to reach LD50 in TS-treated animals. TS effect on survival, though, could be observed with several virus doses (1 X 10(3) to 1 X 10(6) PFU) (p less than 0.001). A significant effect on survival was also observed with outbred ICR mice (p less than 0.005). Serum interferon (IFN) levels in the Mengo virus-infected mice were relatively low (average peak 300 U/ml), but were significantly increased (two- to ninefold) in the TS-treated mice. Peak serum levels were reached earlier in TS than in control animals (24 hr and 72 hr, respectively). Both acid-labile and acid-stable type I IFN production were augmented by TS in the Mengo virus-infected mice. Natural killer activity was also enhanced by TS, in particular on the second day after virus inoculation. In addition, MP-virus was used as a second, unrelated virus challenge. This virus caused a nonlethal infection, with relatively high levels of serum IFN (average peak 10,000 U/ml). TS increased IFN levels (two- to eight-fold) also in this challenge system. In conclusion, TS causes a nonspecific enhancement of endogenous production of IFN and has a significant effect on the survival of lethally infected mice. The data indicate a potential application of thymic factors for the treatment of viral infections.     

Pathogenesis of Neonatal Herpes Simplex 2 Disease in a Mouse Model Is Dependent on Entry Receptor Expression and Route of Inoculation

Herpes simplex virus (HSV) pathogenesis in mice differs based on availability of the principal entry receptors herpesvirus entry mediator (HVEM) and nectin-1 in a manner dependent upon route of inoculation. After intravaginal or intracranial inoculation of adult mice, nectin-1 is a major mediator of neurologic disease, while the absence of either receptor attenuates disease after ocular infection. We tested the importance of receptor availability and route of infection on disease in mouse models of neonatal HSV. We infected 7-day-old mice lacking neither or one principal HSV receptor or both principal HSV receptors with HSV-2 via a peripheral route (intranasal), via a systemic route (intraperitoneal), or by inoculation directly into the central nervous system (intracranial). Mortality, neurologic disease, and visceral dissemination of virus were significantly attenuated in nectin-1 knockout mice compared with HVEM knockout or wild-type mice after intranasal inoculation. Mice lacking both entry receptors (double-knockout mice) showed no evidence of disease after inoculation by any route. Nectin-1 knockout mice had delayed mortality after intraperitoneal inoculation relative to wild-type and HVEM knockout mice, but virus was able to spread to the brain and viscera in all genotypes except double-knockout mice. Unlike in adult mice, HVEM was sufficient to mediate disease in neonatal mice after direct intracranial inoculation, and the absence of HVEM delayed time to mortality relative to that of wild-type mice. Additionally, in wild-type neonatal mice inoculated intracranially, HSV antigen did not primarily colocalize with NeuN-positive neurons. Our results suggest that differences in receptor expression between adults and newborns may partially explain differences in susceptibility to HSV-2.     

Enhancement of DTH response by cholera toxin B subunit inoculated intranasally together with influenza HA vaccine

The effects of B subunit of cholera toxin (CTB) on delayed-type hypersensitivity (DTH) response to influenza vaccine derived from influenza virus A/PR/8/34 (PR-8, HlNl) virus were investigated in B10 mice that were immunized intranasally with both influenza vaccine and CTB. The result showed that intranasal inoculation of this combination augmented DTH response to influenza vaccine, which reached its peak 6 days after inoculation, and also induced accelerated DTH response upon a second inoculation of influenza vaccine alone 4 weeks later, that the cross-reactive DTH response to PR-8 vaccine was elicited by the injection of the different influenza A-type virus vaccine into the footpad of the vaccinated mice, but was not by influenza B-type virus vaccine, that the DTH-mediating T cells were detected selectively in the lungs of mice that received the nasal inoculation of the vaccine and CTB together, but that subcutaneous inoculation of this combination failed to induce DTH-mediating T cells in the lungs. These results, together with the previous papers (Tamura et al, Vaccine 7: 257-262; 314-320, 1989), suggest that CTB could augment both humoral and DTH responses against influenza vaccine in the respiratory mucosal tract.