Intranasal vaccinations with the trans-sialidase antigen plus CpG Adjuvant induce mucosal immunity protective against conjunctival Trypanosoma cruzi challenges.

Trypanosoma cruzi is an intracellular protozoan parasite capable of infecting through mucosal surfaces. Our laboratory has previously elucidated the anatomical routes of infection after both conjunctival and gastric challenge in mice. We have shown that chronically infected mice develop strong immune responses capable of protecting against subsequent rechallenge with virulent parasites through gastric, conjunctival, and systemic routes of infection. We have also shown that intranasal immunizations with the unique T. cruzi trans-sialidase (TS) antigen protect against gastric and systemic T. cruzi challenge. In the current work we have investigated the ability of purified TS adjuvanted with CpG-containing oligonucleotides to induce immunity against conjunctival T. cruzi challenge. We confirm that intranasal vaccinations with TS plus CpG induce TS-specific T-cell and secretory IgA responses. TS-specific secretory IgA was detectable in the tears of vaccinated mice, the initial body fluid that contacts the parasite during infectious conjunctival exposures. We further show that intranasal vaccinations with TS plus CpG protect against conjunctival T. cruzi challenge, limiting local parasite replication at the site of mucosal invasion and systemic parasite dissemination. We also provide the first direct evidence that mucosal antibodies induced by intranasal TS vaccination can inhibit parasite invasion.

Anatomical route of invasion and protective mucosal immunity in Trypanosoma cruzi conjunctival infection.

Trypanosoma cruzi is a protozoan parasite that can initiate mucosal infection after conjunctival exposure. The anatomical route of T. cruzi invasion and spread after conjunctival parasite contamination remains poorly characterized. In the present work we have identified the sites of initial invasion and replication after contaminative conjunctival challenges with T. cruzi metacyclic trypomastigotes using a combination of immunohistochemical and real-time PCR confirmatory techniques in 56 mice between 3 and 14 days after challenge. Our results demonstrate that the predominant route of infection involves drainage of parasites through the nasolacrimal duct into the nasal cavity. Initial parasite invasion occurs within the ductal and respiratory epithelia. After successive waves of intracellular replication and cell-to-cell spread, parasites drain via local lymphatic channels to lymph nodes and then disseminate through the blood to distant tissues. This model of conjunctival challenge was used to identify immune responses associated with protection against mucosal infection. Preceding mucosal infection induces mucosal immunity, resulting in at least 50-fold reductions in recoverable tissue parasite DNA in immune mice compared to controls 10 days after conjunctival challenge (P < 0.05). Antigen-specific gamma interferon production by T cells was increased at least 100-fold in cells harvested from immune mice (P < 0.05). Mucosal secretions containing T. cruzi-specific secretory immunoglobulin A harvested from immune mice were shown to protect against mucosal parasite infection (P < 0.05), demonstrating that mucosal antibodies can play a role in T. cruzi immunity. This model provides an important tool for detailed studies of mucosal immunity necessary for the development of mucosal vaccines.

Differential interleukin-8 and nitric oxide production in epithelial cells induced by mucosally invasive and noninvasive Trypanosoma cruzi trypomastigotes.

Trypanosoma cruzi metacyclic trypomastigotes (MT), but not blood form trypomastigotes (BFT), are highly mucosally infective. We investigated the abilities of MT and BFT to induce inflammation and/or intracellular killing activity within mucosal epithelia. BFT, but not MT, induced marked increases in interleukin-8, GRO-alpha, MCP-1, and nitric oxide production in HeLa and AGS cells, despite similar infectivities. MT may avoid induction of inflammation as an important biological mechanism facilitating mucosal invasion.

Type 1 immunity provides optimal protection against both mucosal and systemic Trypanosoma cruzi challenges.

Chagas' disease results from infection with Trypanosoma cruzi, a protozoan parasite that establishes systemic intracellular infection after mucosal invasion. We hypothesized that ideal vaccines for mucosally invasive, intracellular pathogens like T. cruzi should induce mucosal type 2 immunity for optimal induction of protective secretory immunoglobulin A (IgA) and systemic type 1 immunity protective against intracellular replication. However, differential mucosal and systemic immune memory could be difficult to induce because of reciprocal inhibitory actions between type 1 and type 2 responses. To test our hypotheses, we investigated the protective effects of type 1 and type 2 biased vaccines against mucosal and systemic T. cruzi challenges. Intranasal vaccinations were given with recombinant interleukin-12 (IL-12)- and IL-4-neutralizing antibody (Ab) for type 1 immune bias, or recombinant IL-4 and gamma interferon-neutralizing Ab for type 2 immune bias. Cytokine RNA and protein studies confirmed that highly polarized memory immune responses were induced by our vaccination protocols. Survival after virulent subcutaneous T. cruzi challenge was used to assess systemic protection. Mucosal protection was assessed by measuring the relative inhibition of parasite replication in mucosal tissues early after oral T. cruzi challenge, using both PCR and quantitative culture techniques. As expected, only type 1 responses protected against systemic challenges (P < 0.01). However, contrary to our original hypothesis, type 1 responses optimally protected against mucosal challenges as well (P < 0.05). Type 1 and type 2 biased vaccines induced similar secretory IgA responses. We conclude that future vaccines for T. cruzi and possibly other mucosally invasive, intracellular pathogens should induce both mucosal and systemic type 1 immunity.

Involvement of CD4(+) Th1 cells in systemic immunity protective against primary and secondary challenges with Trypanosoma cruzi.

In general, gamma interferon (IFN-gamma)-producing CD4(+) Th1 cells are important for the immunological control of intracellular pathogens. We previously demonstrated an association between parasite-specific induction of IFN-gamma responses and resistance to the intracellular protozoan Trypanosoma cruzi. To investigate a potential causal relationship between Th1 responses and T. cruzi resistance, we studied the ability of Th1 cells to protect susceptible BALB/c mice against virulent parasite challenges. We developed immunization protocols capable of inducing polarized Th1 and Th2 responses in vivo. Induction of parasite-specific Th1 responses, but not Th2 responses, protected BALB/c mice against virulent T. cruzi challenges. We generated T. cruzi-specific CD4(+) Th1 and Th2 cell lines from BALB/c mice that were activated by infected macrophages to produce their corresponding cytokine response profiles. Th1 cells, but not Th2 cells, induced nitric oxide production and inhibited intracellular parasite replication in T. cruzi-infected macrophages. Despite the ability to inhibit parasite replication in vitro, Th1 cells alone could not adoptively transfer protection against T. cruzi to SCID mice. In addition, despite the fact that the adoptive transfer of CD4(+) T lymphocytes was shown to be necessary for the development of immunity protective against primary T. cruzi infection in our SCID mouse model, protective secondary effector functions could be transferred to SCID mice from memory-immune BALB/c mice in the absence of CD4(+) T lymphocytes. These results indicate that, although CD4(+) Th1 cells can directly inhibit intracellular parasite replication, a more important role for these cells in T. cruzi systemic immunity may be to provide helper activity for the development of other effector functions protective in vivo.

Influenza virus vaccination of patients with chronic lung disease.

STUDY OBJECTIVES: To evaluate the safety of, and mucosal and systemic immune responses induced by two influenza virus vaccine regimens in subjects with COPD. DESIGN: Single-center, blinded, randomized, prospective clinical trial evaluating two vaccine regimens. SETTING: Outpatient clinics of St. Louis Department of Veterans Affairs Medical Center. PARTICIPANTS: Volunteers (age range, 42 to 88 years) had preexisting COPD with severe obstruction to airflow on average, were male, and were not receiving immunosuppressive medication. INTERVENTIONS: Twenty-nine volunteers were randomly assigned to receive either bivalent live attenuated influenza A virus vaccine (CAV) or saline solution placebo intranasally. All subjects also received an i.m. injection of trivalent inactivated influenza virus vaccine (TVV) simultaneously. MEASUREMENTS AND RESULTS: Clinical status and pulmonary function measured by spirometry did not change significantly after vaccination. Using hemagglutinins (H1 and H3 HA) which more closely resembled those in CAV, mean levels of anti-HA immunoglobulin A (IgA) antibodies in nasal washings increased significantly after vaccination with CAV and TVV compared to prevaccination, but they did not increase significantly after TVV and intranasal placebo. Mean levels of influenza A virus-stimulated interleukin-2 and -4 produced by peripheral blood mononuclear cells in vitro increased significantly after administration of the combination vaccine regimen and to a lesser extent after TVV and intranasal placebo compared to respective prevaccination levels. The timing of the cytokine response appeared different following CAV and TVV compared to TVV and intranasal placebo. CONCLUSIONS: Intranasally administered CAV was safe when given with i.m. administered TVV and there may be an immunologic advantage to administration of the combination vaccine regimen compared to TVV with intranasal placebo.

Induction of mucosal antibodies by live attenuated and inactivated influenza virus vaccines in the chronically ill elderly.

Induction of local antibody responses to influenza A virus hemagglutinin by coadministration of two vaccines was investigated. Fifty elderly nursing home residents received inactivated trivalent influenza virus vaccine intramuscularly and simultaneously were randomized to receive either bivalent live attenuated influenza A virus vaccine or saline placebo intranasally in a blinded fashion. More significant increases in anti-H1 and -H3 IgA antibodies were detectable in nasal wash specimens of subjects who received live attenuated virus vaccine than in those who received intranasal placebo. The increased anti-hemagglutinin IgA antibody response was of longer duration in recipients of live attenuated vaccine. The change in antibody titers after vaccination was positively correlated with total blood lymphocyte counts measured before vaccination in both vaccinee groups (P < .05). There was a possible advantage of administering live attenuated with inactivated virus vaccines because of enhanced local antibody responses.