Vaccination of cattle with dextran sulphate-binding Babesia bigemina antigens.

Dextran sulphate-bound Babesia bigemina antigens were used in a preliminary vaccination study and were shown to elicit a protective immune response in cattle. A dextran sulphate-binding fraction of B. bigemina was further subfractionated on a Phenyl Sepharose column to give two fractions--one that strongly bound to the column (bound fraction) and one that did not (unbound fraction). Two groups of cattle were each vaccinated with either the bound or the unbound fraction. These two groups of animals along with a control group were then challenged with B. bigemina-infected erythrocytes. Both groups of vaccinated animals showed considerably lower mean daily parasitaemias as compared to the control group.     

Protection against tuberculosis in Eurasian wild boar vaccinated with heat-inactivated Mycobacterium bovis

Tuberculosis (TB) caused by Mycobacterium bovis and closely related members of the Mycobacterium tuberculosis complex continues to affect humans and animals worldwide and its control requires vaccination of wildlife reservoir species such as Eurasian wild boar (Sus scrofa). Vaccination efforts for TB control in wildlife have been based primarily on oral live BCG formulations. However, this is the first report of the use of oral inactivated vaccines for controlling TB in wildlife. In this study, four groups of 5 wild boar each were vaccinated with inactivated M. bovis by the oral and intramuscular routes, vaccinated with oral BCG or left unvaccinated as controls. All groups were later challenged with a field strain of M. bovis. The results of the IFN-gamma response, serum antibody levels, M. bovis culture, TB lesion scores, and the expression of C3 and MUT genes were compared between these four groups. The results suggested that vaccination with heat-inactivated M. bovis or BCG protect wild boar from TB. These results also encouraged testing combinations of BCG and inactivated M. bovis to vaccinate wild boar against TB. Vaccine formulations using heat-inactivated M. bovis for TB control in wildlife would have the advantage of being environmentally safe and more stable under field conditions when compared to live BCG vaccines. The antibody response and MUT expression levels can help differentiating between vaccinated and infected wild boar and as correlates of protective response in vaccinated animals. These results suggest that vaccine studies in free-living wild boar are now possible to reveal the full potential of protecting against TB using oral M. bovis inactivated and BCG vaccines.     

Ability of T cell subsets and their soluble mediators to modulate the replication of Mycobacterium bovis in bovine macrophages

Peripheral blood mononuclear cells (PBMCs) from cattle vaccinated with Bacillus Calmette-Guerin (BCG) were obtained and expanded in vitro by incubation with purified protein derivative. The ability of these cells to modulate the replication of virulent Mycobacterium bovis in autologous-infected macrophages was compared to cells from non-vaccinated controls. Cells from non-vaccinated animals were shown to confer a significant degree of mycobacteriostatic activity to autologous-infected macrophages. This activity was not inhibited by including a neutralizing antibody versus interferon-gamma (IFN-gamma), and was dependent on direct contact between PBMCs and infected macrophages. Addition of autologous PBMCs from BCG-vaccinated cattle was shown to significantly enhance macrophage resistance to M. bovis, and this increased macrophage resistance was partly abrogated by including a neutralizing antibody to IFN-gamma. Addition of T cells from non-vaccinated animals to infected macrophages was associated with a modest increase in macrophage release of TNF-alpha and nitric oxide, whereas PBMCs from vaccinated animals increased very significantly the release of these factors. Neutralization of nitric oxide (NO), by inclusion of monomethyl-L-arginine, significantly diminished the ability of PBMCs from vaccinated animals to enhance macrophage resistance to M. bovis, but had no impact on the ability of T cells from naive animals to modulate macrophage function. The ability of naive cells to increase macrophage anti-M. bovis activity was largely mediated by CD4+ T cells, whereas both CD4+ T cells and CD8+ T cells conferred macrophage resistance to M. bovis in vaccinated animals. These data highlight the role of IFN-gamma and NO in the immune resistance of cattle to M. bovis.     

Babesia bovis: immunity induced by vaccination with a lipid enriched fraction

A chloroform extract from Babesia bovis-infected erythrocytes was used to vaccinate a group of five naive cattle. Following vaccination, the vaccinates, along with a group of control cattle, were challenged with a virulent heterologous strain of B. bovis. The vaccinates, comparative to the controls, showed delayed as well as decreased parasitaemias. The serological and initial biochemical studies suggested that the immune response was elicited by lipid of babesial origin.     

Efficacy of oral and parenteral routes of Mycobacterium bovis bacille Calmette-Guerin vaccination against experimental bovine tuberculosis in white-tailed deer (Odocoileus virginianus): a feasibility study

We investigated the efficacy of oral and parenteral Mycobacterium bovis bacille Calmette-Guerin Danish strain 1331 (BCG) in its ability to protect white-tailed deer (Odocoileus virginianus) against disease caused by M. bovis infection. Twenty-two white-tailed deer were divided into four groups. One group (n=5) received 10(9) colony-forming units (cfu) BCG via a lipid-formulated oral bait; one group (n=5) received 10(9) cfu BCG in culture directly to the oropharynx, one group (n=6) was vaccinated with 10(6) cfu BCG subcutaneously, and one group served as a control and received culture media directly to the oropharynx (n=6). All animals were challenged 3 mo after vaccination. Five months postchallenge the animals were examined for lesions. Results indicate that both oral forms of BCG and parenterally administered BCG offered significant protection against M. bovis challenge as compared to controls. This study suggests that oral BCG vaccination may be a feasible means of controlling bovine tuberculosis in wild white-tailed deer populations.     

A haemolytic cell-free preparation of Moraxella bovis confers protection against infectious bovine keratoconjunctivitis

Abstract Protection conferred by a cell-free preparation from a haemolytic Moraxella bovis isolate, UQV 148NF, was compared to an equivalent fraction from a non-haemolytic M. bovis isolate, Gordon 26L3, and to a recombinant DNA-derived pili vaccine. Three groups of ten calves were vaccinated twice with one of the three preparations and, together with ten non-vaccinated calves, challenged with virulent M. bovis isolate Dal 2d. Compared to the control group, significant protection was observed in the group receiving the pili vaccine and the group receiving the preparation from haemolytic isolate, UQV 148NF.     

The lysate from bovine erythrocytes infected with Babesia bovis. Analysis of antigens and a report on their immunogenicity when polymerized with glutaraldehyde

A group of Babesia bovis antigens obtained by a lengthy biochemical procedure involving disruption of infected erythrocytes has previously been shown to be highly protective. This study shows that these antigens can be found in a simple lysate of infected erythrocytes. The antigens have been characterized by gel filtration and nitrocellulose transfer and consist of a wide spectrum of molecular sizes. Some of the antigens exist in complex form and are easily dissociated. The lysate was polymerized with glutaraldehyde and injected per se into four splenectomized calves. All the calves produced antibody to B. bovis but did not produce erythrocytic isoantibodies. The vaccinated calves and a control group of four splenectomized calves were challenged with virulent B. bovis. Statistically, the vaccinated group differed significantly in parasitaemia, temperature change and pathophysiological parameters from the control group. All of the control group died whereas two of the vaccinated group survived infection.     

Vaccination of vampire bats using recombinant vaccinia-rabies virus

Adult vampire bats (Desmodus rotundus) were vaccinated by intramuscular, scarification, oral, or aerosol routes (n = 8 in each group) using a vaccinia-rabies glycoprotein recombinant virus. Sera were obtained before and 30 days after vaccination. All animals were then challenged intramuscularly with a lethal dose of rabies virus. Neutralizing antirabies antibodies were measured by rapid fluorescent focus inhibition test (RFFIT). Seroconversion was observed with each of the routes employed, but some aerosol and orally vaccinated animals failed to seroconvert. The highest antibody titers were observed in animals vaccinated by intramuscular and scarification routes. All animals vaccinated by intramuscular, scarification, and oral routes survived the viral challenge, but one of eight vampire bats receiving aerosol vaccination succumbed to the challenge. Of 31 surviving vaccinated and challenged animals, nine lacked detectable antirabies antibodies by RFFIT (five orally and four aerosol immunized animals). In contrast, nine of 10 non-vaccinated control bats succumbed to viral challenge. The surviving control bat had antiviral antibodies 90 days after viral challenge. These results suggest that the recombinant vaccine is an adequate and safe immunogen for bats by all routes tested.     

EIAV弱毒疫苗免疫马外周血单核细胞IL-2表达水平的研究

目的:通过定量监测马传染性贫血病毒(EIAV)弱毒疫苗免疫马外周血单核细胞(PBMC)IL-2表达水平的变化特征,探讨EIAV弱毒疫苗的免疫保护机制。方法:用实时定量RT-PCR技术建立了马外周血PBMCIL-2表达水平的定量检测方法。在不同的时间点定期对4组(疫苗免疫组、健康对照组、强毒攻毒组和EIAV自然感染组)12匹马的外周血PBMCIL-2表达水平进行了检测,同时观察了临床症状及体温变化等指标。疫苗株免疫动物8个月后用强毒攻毒,观察了攻毒前后IL-2表达水平的变化。结果:(1)疫苗免疫马外周血PBMCIL-2的表达量显著高于健康对照组及自然感染组(P<0.01),且免疫后攻毒IL2继续升高,4匹疫苗免疫马均获得完全保护;(2)强毒攻毒对照组IL2表达量随疾病进展波动,发热期明显下降。结论:首次证明EIAV弱毒疫苗可诱导马外周血PBMC表达高水平的IL-2,提示IL-2在疫苗的免疫保护应答中发挥了重要作用;IL-2表达水平还与EIAV感染后的疾病进展密切相关。     

Vaccination with cathepsin L phage-exposed mimotopes,single or in combination,reduce size,fluke burden,egg production and viability in sheep experimentally infected with Fasciola hepatica

Fascioliasis is a worldwide emergent zoonotic disease that significantly constrains the productivity of livestock. In this study, fluke burdens, liver fluke size and biomass, faecal eggs counts, serum levels of hepatic enzymes and immune response were assessed in sheep vaccinated with peptide mimotopes of cathepsin L and infected with metacercariae. A total of 25 sheep were allocated randomly into five groups of five animals each, and experimental groups were immunised with 1 × 10¹³ filamentous phage particles of cathepsin L1 (CL1) (TPWKDKQ), CL2 (YGSCFLR) and mixtures of CL1 + CL2 mimotopes, in combination with Quil A adjuvant, and wild-type M13KE phage in a two-vaccination scheme on weeks 0 and 4. The control group received phosphate-buffered saline. All groups were challenged with 300 metacercariae two weeks after the last immunisation and euthanised 16 weeks later. The CL1 vaccine was estimated to provide 57.58% protection compared with the control group; no effect was observed in animals immunised with CL2 and CL1 + CL2 (33.14% and 11.63%, respectively). However, animals receiving CL2 had a significant reduction in parasite egg output. Vaccinated animals showed a significant reduction in fluke length and width and wet weights. In the CL1 group, there was a significant reduction in the total biomass of parasites recovered. Egg development was divided into seven stages: dead, empty, unembryonated, cell division, eyespot, hatched and hatching. The highest percentage of developmental stages was detected for vaccinated sheep administered CL1 + CL2 with cell division, and the lowest percentage was observed in the hatching stage. Furthermore, a significant difference in all developmental stages was observed between vaccinated animals and the control group (P < 0.01). The levels of anti-phage total IgG in immune sera increased significantly at four weeks after immunisation and were always significantly higher for cathepsin L vaccine group than in the challenged control group. Total IgG was inversely and significantly correlated with worm burden in the CL1 group.     

The irradiation of Babesia bovis. 1. The difference in pathogenicity between irradiated and non-irradiated populations

Babesia bovis parasites attenuated by 35 krads gamma irradiation and parasites not exposed to irradiation, were injected into intact 2-year-old Hereford steers. All five animals receiving non-irradiated blood died but the five animals which received irradiated blood were only mildly affected. Highly significant differences were observed in changes to plasma fibrinogen, serum fibrinogen-like proteins, packed cell volume, partial thromboplastin time, prothrombin time, blood kinins, and plasma kininogen levels in the control animals but non-significant changes in these parameters occurred in the group receiving iradiated blood. Significant changes in the antiplasmin alpha 2M, and the antithrombin levels occurred in control cattle but not in the group receiving irradiated blood. Parasite multiplications rates and maximum parasitaemias were similar in both groups. Irradiation reduced the dose of living parasites from 1 x 10(8) to 2.5 x 10(3), but this was not the reason for the mild reactions. It was concluded that irradiation had selected an avirulent parasite population.     

Efficacy of a Vaccine Formula against Tuberculosis in Cattle

“Test-and-slaughter” has been successful in industrialized countries to control and eradicate tuberculosis from cattle; however, this strategy is too expensive for developing nations, where the prevalence is especially high. Vaccination with the Calmette-Guérin (BCG) strain has been shown to protect against the development of lesions in vaccinated animals: mouse, cattle and wildlife species. In this study, the immune response and the pathology of vaccinated (BCG-prime and BCG prime-CFP-boosted) and unvaccinated (controls) calves were evaluated under experimental settings. A 10⁶ CFU dose of the BCG strain was inoculated subcutaneously on the neck to two groups of ten animas each. Thirty days after vaccination, one of the vaccinated groups was boosted with an M. bovis culture filtrate protein (CFP). Three months after vaccination, the three groups of animals were challenged with 5×10⁵ CFU via intranasal by aerosol with a field strain of M. bovis. The immune response was monitored throughout the study. Protection was assessed based on immune response (IFN-g release) prechallenge, presence of visible lesions in lymph nodes and lungs at slaughter, and presence of bacilli in lymph nodes and lung samples in histological analysis. Vaccinated cattle, either with the BCG alone or with BCG and boosted with CFP showed higher IFN-g response, fewer lesions, and fewer bacilli per lesion than unvaccinated controls after challenge. Animals with low levels of IFN-g postvaccine-prechallenge showed more lesions than animals with high levels. Results from this study support the argument that vaccination could be incorporated into control programs to reduce the incidence of TB in cattle in countries with high prevalence.     

Heparin binding haemagglutin as potential diagnostic marker of Mycobacterium bovis

In this study, we characterized the humoral responses in cattle of Sardinia. The animals were divided into three groups: 1) 28 cattle infected with Mycobacterium bovis; 2) 48 cattle from herds in which foci of infection was notified; 3) 50 cattle from herds that were TB-free. Levels of IgG antibody were measured against the following antigens of M. tuberculosis: Heparin-Binding-Haemagglutin (HBHA), Ag85B, PPE44, and PE_PGRS33 to investigate their potential to diagnose TB in animals. Our results indicated that HBHA is a potential candidate for the development of a serological assay for rapid diagnosis of cattle infected with M. bovis.     

Vaccination with Plasmodium knowlesi AMA1 formulated in the novel adjuvant co-vaccine HT™ protects against blood-stage challenge in rhesus macaques

Plasmodium falciparum apical membrane antigen 1 (PfAMA1) is a leading blood stage vaccine candidate. Plasmodium knowlesi AMA1 (PkAMA1) was produced and purified using similar methodology as for clinical grade PfAMA1 yielding a pure, conformational intact protein. Combined with the adjuvant CoVaccine HT™, PkAMA1 was found to be highly immunogenic in rabbits and the efficacy of the PkAMA1 was subsequently tested in a rhesus macaque blood-stage challenge model. Six rhesus monkeys were vaccinated with PkAMA1 and a control group of 6 were vaccinated with PfAMA1. A total of 50 µg AMA1 was administered intramuscularly three times at 4 week intervals. One of six rhesus monkeys vaccinated with PkAMA1 was able to control parasitaemia, upon blood stage challenge with P. knowlesi H-strain. Four out of the remaining five showed a delay in parasite onset that correlated with functional antibody titres. In the PfAMA1 vaccinated control group, five out of six animals had to be treated with antimalarials 8 days after challenge; one animal did not become patent during the challenge period. Following a rest period, animals were boosted and challenged again. Four of the six rhesus monkeys vaccinated with PkAMA1 were able to control the parasitaemia, one had a delayed onset of parasitaemia and one animal was not protected, while all control animals required treatment. To confirm that the control of parasitaemia was AMA1-related, animals were allowed to recover, boosted and re-challenged with P. knowlesi Nuri strain. All control animals had to be treated with antimalarials by day 8, while five out of six PkAMA1 vaccinated animals were able to control parasitaemia. This study shows that: i) Yeast-expressed PkAMA1 can protect against blood stage challenge; ii) Functional antibody levels as measured by GIA correlated inversely with the day of onset and iii) GIA IC₅₀ values correlated with estimated in vivo growth rates.     

Reduced infection and protection from clinical signs of cerebral neosporosis in C57BL/6 mice vaccinated with recombinant microneme antigen NcMIC1

NcMIC1 is a 460 amino acid Neospora caninum microneme protein implicated in host cell adhesion and invasion processes. In this study, we assessed the potential protectivity of NcMIC1-based vaccination against experimental N. caninum infection in mice, employing both recombinant antigen vaccines and DNA vaccines. Recombinant NcMIC1 (recNcMIC1) was expressed in Escherichia coli as gluthatione-S-transferase-fusion protein. The corresponding NcMIC1 cDNA was cloned into the pcDNA3.1 expression plasmid (pcDNA-MIC1), and expression was checked in transfected Vero cells. Mice (10 animals/group) were vaccinated either with recNcMIC1 antigen suspended in Ribi-adjuvant (3 intraperitoneal injections), pcDNA-NcMIC1 (3 intramuscular injections), or pcDNA-NcMIC1 (twice intramuscularly), followed by 1 intraperitoneal recNcMIC1 antigen boost. Control groups included corresponding treatments with adjuvant, pcDNA3.1 without insert, and PBS (= infection control). All vaccinated and control groups were then challenged intraperitoneally with 2 x 10(6) N. caninum tachyzoites. Animals were inspected daily for a period of 3 wk postinfection (PI). At day 21, all animals were killed and assessed for infection. Before day 21 PI, clinical signs such as walking disorders, rounded back, apathy, and paralysis occurred in infection controls (50% of the mice), pcDNA and adjuvant controls (20% each), and the combined pcDNA-NcMIC1/recNcMIC1-treated group (30%). No clinical symptoms were observed in the recNcMIC1 and pcDNA-NcMIC1 vaccinated groups. All mice were positive for cerebral N. caninum infection as assessed by PCR of brain tissue. However, quantitative real-time PCR revealed that the infection intensity was significantly reduced in the group vaccinated with recNcMIC1 antigen. Immunohistochemistry confirmed these findings. In contrast, the infection intensity was highest in the group vaccinated with the pcDNA-NcMIC1/recNcMIC1 combination, indicating that the sequential application of the DNA vaccine and recombinant antigen had a deleterious effect. Serological analysis showed that only recNcMIC1-immunized animals generated detectable antibody levels recognizing native NcMIC1. Thus, of all protocols applied here, only recNcMIC1 vaccination appears to be suited to reduce cerebral infection in mice challenged with N. caninum tachyzoites.     

Immune modification of the pathogenesis of Streptococcus uberis mastitis in the dairy cow

Abstract Two groups of 4 cows were vaccinated subcutaneously with live Streptococcus uberis strain 0140J or a surface extract derived from the same strain, at 14 days prior to the cessation of lactation (drying off) and at calving. Both groups also received an intramammary administration of the surface extract 7 days after drying off. A third group of unvaccinated animals acted as controls. Following intramammary challenge of two quarters per cow with the vaccine strain, all quarters on control cows and those vaccinated only with surface extract developed clinical mastitis. However, only 12.5% of challenged quarters on cows which were vaccinated with live bacteria developed clinical mastitis. In addition, the numbers of bacteria in the milk following challenge were 10⁵ times higher from the control and extract vaccinated cows than those which received live vaccine. Serum levels of S. uberis specific IgG₂ were elevated in the animals vaccinated with the live organism when compared to that of either extract-vaccinates or controls, whilst S. uberis specific levels of IgG₁ and IgM were similar in all groups throughout the experiment. Specific antibody levels in milk were unaffected by vaccination. Despite increased levels of IgG₂, no increase in opsonic activity was detected in any serum or milk samples. Peripheral blood lymphocytes from animals vaccinated with live organisms showed a considerable increase in proliferative response to S. uberis antigen in vitro when compared with lymphocytes from control and extract-vaccinated animals. These results suggest that neutrophils and specific opsonising antibody may not form the major defence against infection with S. uberis .     

A synthetic bmti n-terminal fragment as antigen in bovine immunoprotection against the tick Boophilus microplus in a pen trial

Cattle tick control remains a serious problem in cattle farms worldwide, due to the limited success achieved with chemicals. Although the use of vaccines for tick control may open possibilities for an integrated approach, the search for other protective antigens is still necessary to improve control. Boophilus microplus is a rich source of BmTIs, serine proteinase inhibitors that are present throughout the tick's lifecycle. The present paper reports a pen trial conducted to evaluate the performance of a synthetic BmTI N-terminal fragment as antigen against the tick in bovines. The trial was conducted with two groups of eight crossbred cattle under controlled infestation: one group was vaccinated with BmTI peptide using saponin as adjuvant and the other was kept as control. Challenge was performed with 15,000 larvae. The specific IgG response was measured by ELISA, with successful outcomes for vaccinated animals. Vaccination resulted in an 18.4% level of efficacy when compared with the control group.     

Resistance of vaccinated mice to typical and atypical strains of Coccidioides immitis

In earlier reports, it was shown that mice and monkeys could be immunized against otherwise lethal challenge doses of Coccidioides immitis arthrospores. The vaccine was composed of Formalin-killed, in vitro grown, endosporulating spherules of C. immitis strain Silveira. In this study, mice were immunized as in the earlier work and then challenged intranasally with arthrospores from seven heterologous strains of C. immitis. Two of these strains were typical of the species, and five were atypical with respect to their cultural characteristics and morphology of microscopic structures. The vaccinated animals were well protected against challenge doses that were lethal to a majority of the control animals, regardless of the strain of fungus employed. The infection ratios among surviving vaccinated and control animals were comparable, but demonstrable lesions were generally smaller and less numerous in the vaccinated groups. It is suggested that these strains are at least immunogenically similar, although not necessarily identical, and that a vaccine prepared from a single strain of C. immitis would be practical for an immunization program.     

Establishing populations of Megasphaera elsdenii YE 34 and Butyrivibrio fibrisolvens YE 44 in the rumen of cattle fed high grain diets

AIM: To determine whether Megasphaera elsdenii YE34 (lactic acid degrader) and Butyrivibrio fibrisolvens YE44 (alternative starch utilizer to Streptococcus bovis) establish viable populations in the rumen of beef cattle rapidly changed from a forage-based to a grain-based diet. METHODS AND RESULTS: Five steers were inoculated with the two bacterial strains (YE34 and YE44) and five served as uninoculated controls. With the exception of one animal in the control group, which developed acidosis, all steers rapidly adapted to the grain-based diet without signs of acidosis (pH decline and accumulation of lactic acid). Bacterial populations of S. bovis, B. fibrisolvens and M. elsdenii were enumerated using real-time Taq nuclease assays. Populations of S. bovis remained constant (except in the acidotic animal) at ca 10(7) cell equivalents (CE) ml-1 throughout the study. Megasphaera elsdenii YE34, was not detectable in animals without grain in the diet, but immediately established in inoculated animals, at 10(6) CE ml-1, and increased 100-fold in the first 4 days following inoculation. Butyrivibrio fibrisolvens, initially present at 10(8) CE ml-1, declined rapidly with the introduction of grain into the diet and was not detectable 8 days after grain introduction. CONCLUSION: Megasphaera elsdenii rapidly establishes a lactic acid-utilizing bacterial population in the rumen of grain-fed cattle 7-10 days earlier than in uninoculated cattle. SIGNIFICANCE AND IMPACT OF THE STUDY: The study has demonstrated that rumen bacterial populations, and in particular the establishment of bacteria inoculated into the rumen for probiotic use, can be monitored by real-time PCR.     

Development of vaccines to control bovine tuberculosis in cattle and relationship to vaccine development for other intracellular pathogens

Vaccination of cattle against bovine tuberculosis could be an important strategy for the control of disease either where there is a wildlife reservoir of Mycobacterium bovis infection or in developing countries where it is not economically feasible to implement a 'test and slaughter' control program. Advances in the understanding of protective immune responses to M. bovis infection in cattle and the advent of new molecular biological techniques, coupled with the sequencing of the M. bovis genome have provided opportunities for the rational development of improved tuberculosis vaccines. A number of new tuberculosis vaccines including attenuated M. bovis strains, killed mycobacteria, protein and DNA vaccines are under development and many are being assessed in cattle. Recent results have revealed several promising vaccine candidates and vaccination strategies. Ways of distinguishing between vaccinated and infected cattle are becoming available and the possibility of new approaches to the eradication of tuberculosis from domestic livestock is discussed. Similarities between the mechanisms of protective immunity against M. bovis and against other intracellular parasites continue to be found and discoveries from vaccine studies on bovine tuberculosis may provide helpful insights into requirements for vaccines against other intracellular pathogens.