Passive transfer of protective immunity to larval Dirofilaria immitis from dogs to BALB/c mice

Protective immunity to larval Dirofilaria immitis has been demonstrated in both the natural host, the dog, and in an experimental host, the mouse. In the present study, sera were collected and pooled from dogs that had been shown to have protective immunity to larval D. immitis. The pooled serum was inoculated into normal BALB/cByJ mice that then were challenged with third-stage larvae (L3) implanted in diffusion chambers. Two weeks postchallenge no significant difference was seen in either parasite survival or growth. Three weeks postchallenge, there was a significant decrease in parasite survival in mice receiving serum from immune dogs. Living larvae recovered at 3 wk postchallenge were significantly shorter than cohorts recovered from control mice. Antibody responses to L3 and forth-stage larvae (L4) surface antigens, to L3 and L4 aqueous soluble antigens, and to an excretory-secretory antigen fraction were measured. Only antibody responses to L3 surface antigens were elevated in the immune serum as compared to controls, thus suggesting a possible role for antibodies with specificity for surface antigens in protective immunity.     

Onchocerca volvulus: immunization of chimpanzees with X-irradiated third-stage (L3) larvae.

To provide a theoretical basis for the potential development of vaccines against Onchocerca volvulus (Ov) a trial has been conducted to assess the protective efficacy of immunization of chimpanzees with X-irradiated L3 larvae. Approximately 1000 larvae were injected at 0, 1, and 7 months. The immunized animals, and unimmunized controls, were then challenged with 250 live L3. In order to provide possibly protective exposure to the immunologically distinct L4 epicuticle, a radiation dose (45 krad) was chosen which preserved about 50% of the molting ability of unirradiated larvae. Despite the presence of a strong immune response to crude adult worm extracts, and to cloned Ov antigens, at the time of challenge little or no significant protection against patent infection was observed: three of four immunized animals developed patent infection as compared to four of four controls. One immunized animal failed to become patent or to manifest the late antibody response to adult worm antigens seen in both subpatent and patent infections in this model, and may have been protected from infection. The implications of these studies for future attempts to immunize against O. volvulus are discussed.     

Immunoaffinity-isolated antigens induce protective immunity against larval Strongyloides stercoralis in mice

The objective of this study was to identify soluble protein antigens that would induce protective immunity against infective-stage larvae (L-3) of Strongyloides stercoralis in mice. Deoxycholate (DOC)-soluble proteins derived from L-3, adsorbed to aluminum hydroxide, induced protective immunity in BALB/c mice. The immunized mice generated parasite-specific IgG that could transfer passive immunity to na?ve animals. The protective antibodies bound to parasite antigens found in the muscles and nerve cords of the L-3. An IgG affinity chromatography column generated with IgG from the sera of DOC-immunized mice was used to purify specific larval antigens. Proteins were eluted from the affinity column with sizes of 80, 75, 61, 57, 43, and 32 kDa. This antigen pool stimulated both proliferation and IL-5 production by splenocytes recovered from mice immunized with live L-3. Vaccination of mice with the immunoaffinity-isolated antigens led to significant protective immunity, with 83% of challenge larvae killed. This study demonstrates that IgG-isolated proteins are candidate antigens for a vaccine against larval S. stercoralis.     

Acanthocheilonema viteae: vaccination of jirds with irradiation-attenuated stage-3 larvae and with exported larval antigens

Jirds (Meriones unguiculatus) were immunized with irradiated (35 krad) stage-3 larvae (L3) of Acanthocheilonema viteae. The induced resistance against homologous challenge infection and the antibody response of the animals were studied. Immunization with 3, 2, or 1 dose of 50 irradiated L3 induced approximately 90% resistance. Immunization with a single dose of only 5 irradiated L3 resulted in 60.8% protection while immunization with a single dose of 25 L3 induced 94.1% protection. The protection induced with 3 doses of 50 irradiated L3 did not decrease significantly during a period of 6 months. Sera of a proportion, but not all resistant jirds, contained antibodies against the surface of vector derived L3 as defined by IFAT. No surface antigens of microfilariae or adult worms were recognized by the sera. Vaccinated animals had antibody responses against antigens in the inner organs of L3 and in the cuticle and reproductive organs of adult worms as shown by IFAT. Immunoblotting with SDS-PAGE-separated L3 antigens and L3-CSN revealed that all sera contained antibodies against two exported antigens of 205 and 68 kDa, and against a nonexported antigen of 18 kDa. The 205-kDa antigen easily degraded into fragments of 165, 140, 125, and 105 kDa which were recognized by resistant jird sera. Various antigens of adult worms, but relatively few antigens of microfilariae, were also recognized. To test the relevance of exported antigens of L3 to resistance, jirds were immunized with L3-CSN together with a mild adjuvant. This immunization induced 67.7% resistance against challenge infection and sera of the immunized animals recognized the 205- and 68-kDa antigens of L3.     

Studies of stage-specific immunity against Trichostrongylus colubriformis in sheep: immunization by normal and truncated infections.

Sheep immunized with multiple normal infections of 30,000 Trichostrongylus colubriformis larvae (T.c. L3) suppressed the fecundity, establishment and survival of adoptively transferred adult worms, showing that these parasites were susceptible to the effects of host immunity. When sheep were immunized by four 'truncated' larval infections of 4, 7 or 10 days' (d) duration with 10(5) T.c. L3, animals given 4 x 4d infections were susceptible to challenge, whereas sheep given 4 x 7d and 4 x 10d infections were significantly protected. A serial analysis of the rejection of T. colubriformis from nine sheep given 5 x 7d infections revealed that the challenge larval infection given intraduodenally was expelled within 3 days after challenge (DAC). However, another five of these sheep only rejected around 50% of transferred adult worms by 21 DAC when compared with control animals. The results indicate that stage-specific antigens produced by early L3 and L4 stages of T. colubriformis effectively immunize sheep against a larval challenge but appear less reliably protective against adult worms.     

Immunization of sheep with larval antigens of Lucilia cuprina

Sheep were immunized with antigens extracted from third-instar larvae of L. cuprina. This procedure produced substantial titres of circulating antibody as measured by solid-phase radioimmunoassay or immunodiffusion or by both techniques. However, immunization did not confer protection against subsequent implant challenge with first-instar larvae. In vitro studies indicated that pooled sera from immunized sheep (mean immunodiffusion titre = 3) significantly reduced larval survival. Antigen specificity and the modulating effects of concomitant humoral responses to larval challenge are discussed in relation to the findings.     

Genetic control of murine immune responses to larval Dirofilaria immitis

Previous studies have demonstrated that BALB/c mice, immunized against infection with Dirofilaria immitis, were capable of killing a significant percentage of challenge larvae found within diffusion chambers. The percentage of larvae killed by immunized mice was, however, less than in immunized dogs and unlike immunized dogs, mice were unable to retard the development of the surviving larvae. The objective of the present study was to test 3 inbred strains of mice to determine whether a higher level of protective immunity would develop in these hosts and if larval growth retardation would occur. DBA/2J and C57BL/6J mice and their F1 hybrids B6D2F1/J were used in these studies; it was determined that there were differences in susceptibility among the 3 strains but no difference in ability to eliminate larvae from challenge infections. Growth retardation was seen in larvae recovered from immunized DBA/2J and C57BL/6J mice but not in B6D2F1/J. No difference was noted between immune and control mice in the cell types found in the diffusion chambers. The predominant cell types seen were mononuclear macrophages, multinucleate syncytial cells, and neutrophils. Antibody responses to soluble third- and fourth-stage larval antigens and larval excretory/secretory antigens were measured. Although antibodies to all 3 antigen groups were found in higher concentrations in immunized mice than in their respective controls, only antibody responses to soluble L-3 antigens provided a clear correlation with protective immunity.     

Immunogenicity of a chemical typhus vaccine and of a corpuscular radioantigen obtained from Rickettsia prowazekii

The protective activity of chemical typhus vaccine and R. prowazekii corpuscular radioantigen (CRA) was studied. Guinea pigs were immunized with doses of 32 and 48 antigenic units. Antibody production was assayed in the complement fixation test. On days 7, 15, 21, 30 and 60 after immunization the animals were challenged with R. prowazekii introduced in an amount of 10(5) minimum embryonal infective doses (MEID). On day 30 some of the animals were challenged with 10(3) MEID of R. typhi. The results demonstrated that both preparations were highly immunogenic and capable of protecting most of the animals from 10(5) MEID of R. prowazekii. Immunity developed earlier after immunization with CRA. The guinea pigs immunized with CRA, purified in percoll density gradient, and challenged with 10(3) MEID of R. typhi on day 30 showed a high level of cross immunity. In all control animals high fever and periorchitis were observed.     

Hymenolepis nana: intestinal tissue phase in actively immunized mice.

We investigated the fate of the intestinal cestode Hymenolepis nana in immunized mice. Immunity was induced by infection with the parasite eggs. These immunized animals and unimmunized controls were then challenged with 50,000 H. nana eggs. The mice were killed 4 to 90 hr after challenge, and H. nana in the intestinal tissue were counted. At 4 hr after challenge the unimmunized and immunized animals had approximately equal numbers of oncospheres. By 12 hr there were fewer parasites in the immunized than in the unimmunized animals. At 90 hr, no H. nana were seen in the immunized mice, whereas in the unimmunized animals the median number of cysticercoids was more than 1,000. It appears, therefore, that in mice well immunized to H. nana by infection, challenge oncospheres can burrow into the intestinal tissue before they are killed. The reduced number of oncospheres in the immunized mice 12 hr after challenge, and the accumulation of eosinophils near individual oncospheres still present, indicate that an immune response to the parasite was taking place. Absence of a lymphocyte infiltration near any of the oncospheres suggests that the mechanism of immunity was not lymphocyte mediated; thus, the histopathology of the reaction is consistent with that of humoral immunity.     

Resistance to Streptococcus pneumoniae is induced by a phosphocholine-protein conjugate

Previous studies demonstrated that naturally occurring antibodies to the pneumococcal cell wall hapten phosphocholine (PC) are important for the survival of mice against infection with Streptococcus pneumoniae, and that passively administered hybridoma antibody to PC results in added resistance. To determine if a PC-protein conjugate could elicit protective levels of anti-PC antibody, mice were immunized with PC-keyhole limpet hemocyanin (KLH) and tested for their ability to resist challenge with virulent S. pneumoniae. PC-KLH-immunized mice were observed to be resistant to 10- to 1000-fold more organisms than unimmunized control animals. The levels of protection were comparable to those induced with capsular polysaccharide antigens, but had the advantage of not being type-specific; immunization with PC-KLH protected mice against both type 1 and type 3 organisms. The induced immunity appeared to be antibody-mediated; it could be passively transferred with immune serum, and absorption of the immune serum with PC-Sepharose removed its protective capacity. Anti-PC antibodies in the serum of immunized mice were primarily IgM and IgG3 and possessed predominantly the T15 idiotype. Antibodies with these particular isotypes and this idiotype also arise after immunization with heat-killed rough pneumococci and recently were shown to be important in the resistance of mice to pneumococcal infection.     

Immunoprophylaxis against Fasciola hepatica in rabbits using a recombinant Fh15 fatty acid-binding protein.

Previous studies of ours have demonstrated that a recombinant protein (Fh15) related to fatty acid-binding proteins did not induce significant protection in rabbits challenged 2 or 4 wk postimmunization over nonimmunized controls. In the current study, rabbits were immunized with Fh15 and challenged with Fasciola hepatica metacercariae 12 and 20 wk later. In the current study in which longer lag periods for challenge infection after the second immunization were used, worm burden reductions compared to adjuvant controls were a significant 43% and 76%, respectively. Importantly, rabbits immunized with Fh15 had significant numbers of immature flukes, 66% in the 12-wk period and 84% in the 20-wk lag period as compared to controls. In addition, liver lesions were clearly diminished in the vaccinated rabbits. Enzyme-linked immunosorbent assay absorbance values showed that immunized rabbits developed high antibody levels to Fh15 from 8 wk after the first immunization and did not increase after challenge. These results suggest that a recombinant F. hepatica molecule related to fatty acid-binding proteins induces protective (worm burden reductions), anti-fecundity (immature flukes), and anti-pathology (less liver lesions) effects in rabbits and may serve as a model for the immunoprophylaxis of fascioliasis.     

A comparison of the immune response induced by DNA or by an inactivated vaccine against tick-borne encephalitis

BALB/c mice were immunized with recombinant plasmid DNA pSVK3-ENS1 and pcDNAI-NS3 containing, respectively, genes E-NS1 and NS3 of tick-borne encephalitis (TBE) virus. Antibodies to TBE virus proteins were detected in the blood sera of the immunized animals by the method of the enzyme immunoassay. Though the titers of virus-specific antibodies in the sera of mice immunized with protein vaccines exceeded those registered after immunization with DNA vaccines, essential protective immunity was observed after the use of both vaccines.     

Respiratory syncytial virus (RSV) F, G, M2 (22K), and N proteins each induce resistance to RSV challenge, but resistance induced by M2 and N proteins is relatively short-lived.

The ability of recombinant vaccinia viruses that separately encoded 9 of the 10 known respiratory syncytial virus (RSV) proteins to induce resistance to RSV challenge was studied in BALB/c mice. Resistance was examined at two intervals following vaccination to examine early (day 9) as well as late (day 28) immunity. BALB/c mice were inoculated simultaneously by the intranasal and intraperitoneal routes with a recombinant vaccinia virus encoding one of the following RSV proteins: F, G, N, P, SH, M, 1B, 1C, or M2 (22K). A parainfluenza virus type 3 HN protein recombinant (Vac-HN) served as a negative control. One half of the mice were challenged with RSV intranasally on day 9, and the remaining animals were challenged on day 28 postvaccination. Mice previously immunized by infection with RSV, Vac-F, or Vac-G were completely or almost completely resistant to RSV challenge on both days. In contrast, immunization with Vac-HN, -P, -SH, -M, -1B, or -1C did not induce detectable resistance to RSV challenge. Mice previously infected with Vac-M2 or Vac-N exhibited significant but not complete resistance on day 9. However, in both cases resistance had largely waned by day 28 and was detectable only in mice immunized with Vac-M2. These results demonstrate that F and G proteins expressed by recombinant vaccinia viruses are the most effective RSV protective antigens. This study also suggests that RSV vaccines need only contain the F and G glycoproteins, because the immunity conferred by the other proteins is less effective and appears to wane rapidly with time.     

Do sugar residues contribute to the antigenic determinants responsible for protection and/or abolition of protection in Leishmania-infected BALB/c mice?

Intravenous inoculation of irradiated virulent promastigotes of Leishmania mexicana, or intradermal inoculation of avirulent temperature-sensitive clones of the same parasite, can protect BALB/c mice from progressive disease when challenged with parental organisms. However, if animals are prechallenged s.c. with irradiated parental parasites before (or shortly after, i.e., within 10 days) any other immunization regime is used, the s.c. challenge effectively suppresses development of protective immunity. The role of N-linked sugars on promastigotes in providing the determinants responsible for protection and/or suppression of protection in these assays has been examined. Growth of parasite in 2 micrograms/ml tunicamycin has no effect on incorporation of [3H]leucine but decreases incorporation of [3H]mannose by some 40 to 50%. Such tunicamycin-treated avirulent clones or irradiated virulent organisms are now unable to induce a protective response. However, tunicamycin-treated parental parasites given s.c. could still suppress the protection seen when irradiated parasites were given i.v. as immunogen. Tunicamycin-treated avirulent organisms given s.c. subcutaneously unlike the untreated avirulent clones, now also caused suppression of protection. These data suggest that the determinants responsible for development of protective immunity to L. mexicana in BALB/c mice are dependent on N-linked glycoproteins for their expression, unlike the determinants responsible for suppression of that protection. By using swainsonine as an alternative inhibitor of N-linked glycoprotein synthesis, the data additionally suggest that no complex processing of N-linked sugars takes place to reveal the immunogenic determinants in this system.     

Vaccination against cutaneous leishmaniasis in a murine model. I. Induction of protective immunity with a soluble extract of promastigotes

BALB/c mice can be protected against a fatal Leishmania major infection by immunization with whole radio-attenuated promastigotes; however, neither the antigens responsible for protection nor the protective immunologic mechanisms have been defined. In this study, the ability of promastigote fractions to elicit similar immunity to that obtained with whole organisms, and the immune responses associated with such protection were analyzed. Intraperitoneal immunization with a soluble, membrane-free parasite extract was found to induce protection against L. major challenge equal to that obtained with whole organisms. Induction of immunity (89% protection in seven experiments) was most effective with 100 micrograms of the soluble leishmanial antigen (SLA) and required concomitant injection of the bacterial adjuvant, Corynebacterium parvum (CP), followed by an i.p. boost of SLA alone 1 wk later. Vaccinated animals exhibited Leishmania-specific cell-mediated immunity, as assessed both by lymphocyte transformation and the production of macrophage-activating factors (MAF). In addition, although SLA + CP-immunized mice failed to exhibit delayed-type hypersensitivity (DTH) before challenge, splenic lymphocytes from these mice could transfer a local DTH reaction to naive recipients. Immunization also induced the production of antibodies against two major metabolically labeled proteins of m.w. 30,000 and 53,000, but failed to stimulate a detectable humoral response against promastigote surface antigens. Thus, these experiments demonstrate that vaccine-induced immunity against cutaneous leishmaniasis is strongly associated with the induction of cell-mediated immunity, but does not require the development of an antibody response to promastigote surface antigens. In addition, these studies establish the feasibility of employing soluble, nonmembrane-derived parasite material as a source of protective immunogens.     

Th1-Mediated Immunity against Helicobacter pylori Can Compensate for Lack of Th17 Cells and Can Protect Mice in the Absence of Immunization

Helicobacter pylori (H. pylori) infection can be significantly reduced by immunization in mice. Th17 cells play an essential role in the protective immune response. Th1 immunity has also been demonstrated to play a role in the protective immune response and can compensate in the absence of IL-17. To further address the potential of Th1 immunity, we investigated the efficacy of immunization in mice deficient in IL-23p19, a cytokine that promotes Th17 cell development. We also examined the course of Helicobacter infection in unimmunized mice treated with Th1 promoting cytokine IL-12. C57BL/6, IL-12 p35 KO, and IL-23 p19 KO mice were immunized and challenged with H. pylori. Protective immunity was evaluated by CFU determination and QPCR on gastric biopsies. Gastric and splenic IL-17 and IFNγ levels were determined by PCR or by ELISA. Balb/c mice were infected with H. felis and treated with IL-12 therapy and the resulting gastric bacterial load and inflammatory response were assessed by histologic evaluation. Vaccine induced reductions in bacterial load that were comparable to wild type mice were observed in both IL-12 p35 and IL-23 p19 KO mice. In the absence of IL-23 p19, IL-17 levels remained low but IFNγ levels increased significantly in both immunized challenged and unimmunized/challenged mice. Additionally, treatment of H. felis-infected Balb/c mice with IL-12 resulted in increased gastric inflammation and the eradication of bacteria in most mice. These data suggest that Th1 immunity can compensate for the lack of IL-23 mediated Th17 responses, and that protective Th1 immunity can be induced in the absence of immunization through cytokine therapy of the infected host.     

Prophylactic immunization against experimental leishmaniasis. VI. Comparison of protective and disease-promoting T cells

In previous studies, we reported that mice immunized i.v. with lethally irradiated Leishmania major promastigotes developed substantial resistance to a subsequent L. major infection. However, such protection could be totally suppressed by prior s.c. injection with the same antigens. Both the protective immunity and the inhibition of its induction could be adoptively transferred with specific Lyt-2- T cells. Here, we present evidence showing that protection and disease promotion resulting from i.v. or s.c. immunization, respectively, are mediated by functionally distinct subsets of T cells. In a series of titration experiments, it was found that freshly isolated T cells derived from prophylactically i.v. immunized BALB/c mice were either protective (greater than 10(7) cells/recipient) or ineffective (less than 10(7) cells/recipient). No exacerbation of disease was observed at any dose. Conversely, T cells from mice immunized s.c. either accelerated disease development and inhibited protective immunization (greater than 10(7) cells/recipient) or had no effect (less than 10(7) cells/recipient). No protection was observed at any dose tested. In mixed transfer experiments, increasing numbers of T cells from s.c. immunized donors progressively inhibited the protective effect of T cells from i.v. immunized donors. Supernatant of T cell cultures from protectively immunized donors contained substantial macrophage-activating factor whereas such activity was not detectable in the supernatant of T cell culture from s.c. immunized donors. Analysis by flow cytometry showed that the spleen and lymph nodes of normal, i.v., or s.c. immunized BALB/c mice contained similar ratios of L3T4+ cells and Lyt-2+ cells.     

Role of L3T4+ and Lyt-2+ T cell subsets in protective immune responses of mice against infection with a low or high virulent strain of Toxoplasma gondii

In order to elucidate the role of T cell subsets in protective immunity against infection with high virulent and low virulent strains of Toxoplasma gondii, monoclonal antibodies specific for T cell subsets were injected into mice before immunization or challenge infection. Treatment of mice with monoclonal antibody to either L3T4+ or Lyt-2+ T cells before they were immunized with Toxoplasma cell homogenate prepared from high virulent RH strain tachyzoites markedly reduced survival after mice were challenged with low virulent bradyzoites of the Beverley strain. Thus, induction of protective immunity against bradyzoites of the Beverley strain requires the presence of both L3T4+ and Lyt-2+ T cells. In contrast, mice injected with living bradyzoites of the low virulent Beverley strain after immunization with Toxoplasma cell homogenate acquired protective immunity against high virulent tachyzoites of the RH strain. Lyt-2+ T cells alone appear to be final effector cells for protection against the challenge with high virulent RH strain tachyzoites, since treatment of the bradyzoite-immune mice with anti-Lyt-2 antibody, but not anti-L3T4 antibody, before challenge significantly increased mortality.     

Experimental infections with African trypanosomes: IV. Immunization of cattle with gamma-irradiated Trypanosoma rhodesiense

Cattle immunized with gamma-irradiated Trypanosoma rhodesiense were protected against a challenge inoculation of unirradiated trypanosomes given 1 wk later. These animals were still resistant when rechallenged after 8 mo and some residual immunity persisted for 14 mo after immunization. Animals which had undergone self-cure after infection were resistant when challenged 14 mo later. No resistance was observed in animals challenged with a heterologous strain of T. rhodesiense. The infections observed in unprotected bovines were of a mild nature. Leukopenia and fever were common early in the course of infection, but no anemia was evident at any time.     

The effect of adjuvants on the efficacy of a recombinant herpes simplex virus glycoprotein vaccine

A recombinant, truncated HSV type 1 glycoprotein D secreted by Chinese hamster ovary cells (rgD1) was used to compare the ability of several adjuvants to stimulate protective immunity in guinea pigs. Adjuvants tested included CFA, aluminum hydroxide (alum), a lipophilic derivative of muramyl tripeptide (MTP-PE), and a muramyl dipeptide (MDP) covalently conjugated to rgD1. Animals were immunized three times with rgD1 plus the various adjuvants and antibody titers were determined by ELISA. Four weeks after the last immunization, the animals were challenged intravaginally with HSV type 2 and were monitored daily for clinical signs of disease, including frequency and severity of herpetic lesions, incidence of urinary retention, and mortality during the 14-day post-challenge observation period. Animals immunized in the foot-pad with rgD1 formulated with CFA showed the highest antibody titers. Animals immunized in the footpad with rgD1 using MTP-PE in a 4% squalene formulation, alum, or rgD1 conjugated to MDP showed mean antibody titers that were 57, 16, and 13% of the CFA titers, respectively. Immunization with rgD1 plus MTP-PE, alum, or rgD1-MDP conjugate by the i.m. route elicited lower antibody titers than the footpad route of immunization. Results of the viral challenge indicated that clinical symptoms of the groups immunized with rgD1 with CFA or MTP-PE as adjuvant were similar in magnitude and were markedly reduced compared with unimmunized control groups. Animals immunized with rgD1 combined with alum or rgD1-MDP conjugate showed clinical symptoms significantly more severe than the CFA or MTP-PE groups. The protective immunity observed after i.m. immunization of animals with rgD1 and MTP-PE was only slightly lower than animals immunized with the same Ag-adjuvant combination in the footpad. The results indicate that MTP-PE is an effective adjuvant for the recombinant herpes gD vaccine.