An engineered anti-idiotypic antibody-derived killer peptide (KP) early activates swine inflammatory monocytes,CD3+CD16+ natural killer T cells and CD4+CD8α+ double positive CD8β+ cytotoxic T lymphocytes associated with TNF-α and IFN-γ secretion

This study evaluated the early modulation of the phenotype and cytokine secretion in swine immune cells treated with an engineered killer peptide (KP) based on an anti-idiotypic antibody functionally mimicking a yeast killer toxin. The influence of KP on specific immunity was investigated using porcine reproductive and respiratory syndrome virus (PRRSV) and porcine circovirus type 2 (PCV2) as ex vivo antigens. Peripheral blood mononuclear cells (PBMC) from healthy pigs were stimulated with KP and with a scramble peptide for 20 min, 1, 4 and 20 h or kept unstimulated. The cells were analyzed using flow cytometry and ELISA. The same time-periods were used for KP pre-incubation/co-incubation to determine the effect on virus-recalled interferon-gamma (IFN-γ) secreting cell (SC) frequencies and single cell IFN-γ productivity using ELISPOT.KP induced an early dose-dependent shift to pro-inflammatory CD172α⁺CD14^+high monocytes and an increase of CD3⁺CD16⁺ natural killer (NK) T cells. KP triggered CD8α and CD8β expression on classical CD4⁻CD8αβ⁺ cytotoxic T lymphocytes (CTL) and double positive (DP) CD4⁺CD8α⁺ Th memory cells (CD4⁺CD8α^+low CD8β^+low). A fraction of DP cells also expressed high levels of CD8α. The two identified DP CD4⁺CD8α^+high CD8β^+low/+high CTL subsets were associated with tumor necrosis factor alpha (TNF-α) and IFN-γ secretion. KP markedly boosted the reactivity and cross-reactivity of PRRSV type-1- and PCV2b-specific IFN-γ SC. The results indicate the efficacy of KP in stimulating Th1-biased immunomodulation and support studies of KP as an immunomodulator or vaccine adjuvant.     

Differential interactions of virulent and non-virulent H. parasuis strains with na?ve or swine influenza virus pre-infected dendritic cells

Pigs possess a microbiota in the upper respiratory tract that includes Haemophilus parasuis. Pigs are also considered the reservoir of influenza viruses and infection with this virus commonly results in increased impact of bacterial infections, including those by H. parasuis. However, the mechanisms involved in host innate responses towards H. parasuis and their implications in a co-infection with influenza virus are unknown. Therefore, the ability of a non-virulent H. parasuis serovar 3 (SW114) and a virulent serovar 5 (Nagasaki) strains to interact with porcine bone marrow dendritic cells (poBMDC) and their modulation in a co-infection with swine influenza virus (SwIV) H3N2 was examined. At 1 hour post infection (hpi), SW114 interaction with poBMDC was higher than that of Nagasaki, while at 8 hpi both strains showed similar levels of interaction. The co-infection with H3N2 SwIV and either SW114 or Nagasaki induced higher levels of IL-1β, TNF-α, IL-6, IL-12 and IL-10 compared to mock or H3N2 SwIV infection alone. Moreover, IL-12 and IFN-α secretion differentially increased in cells co-infected with H3N2 SwIV and Nagasaki. These results pave the way for understanding the differences in the interaction of non-virulent and virulent strains of H. parasuis with the swine immune system and their modulation in a viral co-infection.     

Development of γδ T cell subset responses in gnotobiotic pigs infected with human rotaviruses and colonized with probiotic lactobacilli

γδ T cell responses are induced by various viral and bacterial infections. Different γδ T cells contribute to activation and regulation of the inflammatory response and to epithelial repair. How γδ T cells respond to rotavirus infection and how the colonization of probiotics influences the γδ T cell response were unknown. In this study, we evaluated by multicolor flow cytometry the frequencies and distribution of total γδ T cells and three major subsets (CD2-CD8-, CD2+CD8- and CD2+CD8+) in ileum, spleen and blood of gnotobiotic (Gn) pigs at early (3-5 days) and late phases (28 days) after rotavirus infection. The Gn pigs were inoculated with the virulent human rotavirus Wa strain and colonized with a mixture of two strains of probiotics Lactobacillus acidophilus and Lactobacillus reuteri. In na?ve pigs, the highest frequency of total γδ T cells was found in blood, followed by spleen and ileum at the early age (8-10 days old) whereas in older pigs (32 days of age) the highest frequency of total γδ T cells was found in ileum and spleen followed by blood. Rotavirus infection significantly increased frequencies of intestinal total γδ T cells and the putatively regulatory CD2+CD8+ γδ T cell subset and decreased frequencies of the putatively proinflammatory CD8- subsets in ileum, spleen and blood at post-infection days (PID) 3 or 5. The three γδ T cell subsets distributed and responded differently after rotavirus infection and/or lactobacilli colonization. The CD2+CD8+ subset contributed the most to the expansion of total γδ T cells after rotavirus infection in ileum because more than 77% of the total γδ T cells there were CD2+CD8+ cells. There was an additive effect between lactobacilli and rotavirus in inducing total γδ T cell expansion in ileum at PID 5. The overall effect of lactobacilli colonization versus rotavirus infection on frequencies of the CD2+CD8+ γδ T cell subset in ileum was similar; however, rotavirus-infected pigs maintained significantly higher frequencies of CD8- subsets in ileum than lactobacilli-colonized pigs. The dynamic γδ T cell responses suggest that γδ T cell subsets may play important roles in different stages of immune responses after rotavirus infection and probiotic colonization. The knowledge on the kinetics and distribution patterns of γδ T cell subsets in na?ve pigs and after rotavirus infection or lactobacilli colonization provides the foundation for further mechanistic studies of their functions.     

Classical swine fever virus infection modulates serum levels of INF-α, IL-8 and TNF-α in 6-month-old pigs

Several studies have highlighted the important role of cytokines in disease development of classical swine fever virus (CSFV) infection. In the present study, we examined the kinetics of 7 porcine cytokines in serum from pigs infected with 3 different CSFV strains. Based on the clinical picture in 6-month-old Danish pigs, the strains used for inoculation were classified as being of low (Bergen), low to moderate (Eystrup) and moderate to high (Lithuania) virulence. The cytokines interferon-alpha (INF-α), interleukin-8 (IL-8) and tumor necrosis factor-alpha (TNF-α) showed increased levels after CSFV infection with more or less comparable course in the 3 groups. However, the cytokine level peaked with a 2–3 days delay in pigs infected with the low virulent strain compared to those infected with a moderately or highly virulent strain. These findings may indicate that INF-α, IL-8 and TNF-α are involved in the immune response during CSFV infection with strains of different virulence.     

In vitro effects of dexamethasone on bovine CD25+CD4+ and CD25−CD4+ cells

This paper investigates the in vitro effect of dexamethasone on bovine CD25^highCD4⁺, CD25^lowCD4⁺ and CD25⁻CD4⁺ T cells. Only a small percentage of bovine CD25^highCD4⁺ (2–4%) and CD25^lowCD4⁺ (1–2%) cells expressed Foxp3. Dexamethasone caused considerable loss of CD25⁻CD4⁺ cells, but it increased the relative and absolute numbers of CD25^highCD4⁺ and CD25^lowCD4⁺ lymphocytes, while at the same time reducing the percentage of Foxp3⁺ cells within the latter subpopulations. Considering all these, as well as the intrinsically poor Foxp3 expression in bovine CD25⁺CD4⁺, it can be concluded that the drug most probably increased the number of activated non-regulatory CD4⁺ lymphocytes. It has been found that changes in cell number were at least partly caused by proapoptotic effect of the drug on CD25⁻CD4⁺ cells and antiapoptotic effect on CD25^highCD4⁺ and CD25^lowCD4⁺ cells. The results obtained from this study indicate that the involvement of CD4⁺ lymphocytes in producing the anti-inflammatory and immunosuppressive effect of dexamethasone in cattle results from the fact that the drug had a depressive effect on the production of IFN-γ by CD25⁻CD4⁺ cells. Secretion of TGF-β and IL-10 by CD4⁺ lymphocytes was not involved in producing these pharmacological effects, because the drug did not affect production of TGF-β and, paradoxically, it reduced the percentage of IL-10⁺CD4⁺ cells.     

Induction of antibody and interferon-γ production in mice immunized with virus-like particles of swine hepatitis E virus

Virus-like particles (VLPs) composed of the truncated capsid protein of swine hepatitis E virus (HEV) were developed and immune responses of mice immunized with the VLPs were evaluated. IgG titers specific for the capsid protein of swine HEV were significantly higher for all groups of mice immunized with the VLPs than those of the negative control mice. Splenocytes from mice immunized with the VLPs also produced significantly greater quantities of interferon (IFN)-γ than interleukin (IL)-4 and IL-10. These newly developed swine HEV VLPs have the capacity to induce antigen-specific antibody and IFN-γ production in immunized mice.     

Enhanced expression of TGFβ protein in lymphoid organs and lung, but not in serum, of pigs infected with a European field isolate of porcine reproductive and respiratory syndrome virus

Transforming growth factor β (TGFβ) is an immunomodulatory cytokine which is able to modulate the host immune response eliciting an inefficient response against pathogens. In this sense, the role of this cytokine in porcine reproductive and respiratory syndrome (PRRS) has been poorly studied and the reported results are contradictory. Thus, in the present study, the expression of TGFβ was analysed both at tissue (lymphoid organs and lung) and serum level to study its correlation with the expression of PRRS virus (PRRSV). To carry out this study, 32 pigs were inoculated with the European PRRSV field isolate 2982 and sequentially killed from 0 dpi to the end of the study (24 dpi). Blood and tissue samples were collected to determine the expression of PRRSV and TGFβ. PRRSV was detected in inoculated animals from 3 dpi until the end of the study, however TGFβ was not detected in sera from inoculated animals. Contrary, an increase of TGFβ antigen was observed both in the lymphoid organs and in the lung of PRRSV-inoculated pigs when compared with control group. Since TGFβ play a role as an immunomodulatory cytokine of the immune response and also in the differentiation of regulatory T cells (Tregs), the upregulation of the TGFβ at tissue level may play a role in the impairment of the host immune response observed during PRRS, being observed a significant correlation between PRRSV and TGFβ expression at lung level.     

IFN-γ expression and infectivity of Toxoplasma infected tissues are associated with an antibody response against GRA7 in experimentally infected pigs

Toxoplasma gondii, an obligate intracellular parasite, can be transmitted to humans via the consumption of infected meat. However, there are currently no veterinary diagnostic tests available for the screening of animals at slaughter. In the current work, we investigated whether cytokine responses in the blood, and antibody responses against recombinant T. gondii GRA1, GRA7, MIC3 proteins and a chimeric antigen EC2 encoding MIC2-MIC3-SAG1, are associated with the infectivity of porcine tissues after experimental infection with T. gondii. Two weeks after experimental infection of conventional 5-week-old seronegative pigs, an IFN-γ response was detected in the blood, with a kinetic profile that followed the magnitude of the GRA7 antibody response. Antibody responses to GRA1, MIC3 and EC2 were very weak or absent up to 6 weeks post infection. Antibodies against GRA7 occurred in all infected animals and were associated with the presence of the parasite in tissues at euthanasia a few months later, as demonstrated by quantitative real-time PCR and isolation by bio-assay. Remarkably, although brain and heart tissue remained infectious, musculus gastrocnemius and musculus longissimus dorsi were found clear of infectious parasites 6 months after experimental infection. Seropositive response in a GRA7 ELISA indicates a Toxoplasma infection in pigs and is predictive of the presence of infectious cysts in pig heart and brain. This new ELISA is a promising tool to study the prevalence of Toxoplasma infection in pigs. Clearance of the infection in certain pig tissues suggests that the risk assessment of pig meat for human health needs further evaluation.     

Genetic characterization of influenza A viruses circulating in pigs and isolated in north-east Spain during the period 2006–2007

Swine influenza virus is one of the most important pathogens involved in the swine respiratory disease complex. Recent serological surveys showed a high prevalence of swine influenza strains belonging to the H₁N₁, H₁N₂ and H₃N₂ subtypes circulating in pigs in Spain. However, little is known about their genome sequence. Five swine influenza strains were isolated from some unrelated outbreaks occurred during 2006–2007, and their complete genome sequences were determined. Phylogenetic analysis revealed that they belonged to the lineages “Avian-Like” H₁N₁, “Human-Like” H₃N₂, and “Human-Like” H₁N₂, showing tight relationships with early or contemporary strains described in Europe. Notably, one virus of the H₁N₂ subtype showed genetic and antigenic divergence with the European contemporary strains or vaccinal strains of the same subtype, suggesting that some local and divergent clusters of the virus may pass unnoticed in routinary subtyping. Finally, analysis on the entire pattern of genome segments suggested that a second reassortment event could have influenced the evolution of that divergent H₁N₂ strain.     

A cross-sectional study of swine influenza in intensive and extensive farms in the northeastern region of the state of São Paulo,Brazil

Swine influenza (SI) is a seasonal infectious disease highly important to the world pig industry. Loss of daily weight gain, increased costs for the prevention and treatment of secondary infections are the main economic losses associated with the presence of this disease. However, some epidemiological features of SI remain quite unclear. This study focused on assessing the prevalence of swine influenza virus (SIV) infection in intensive and extensive pig herds and associating risk factors. A set of 601 blood samples of five intensive farrow-to-finish farms and 361 blood samples from 56 extensive farms were analyzed using an indirect ELISA kit CIVTEST SUIS INFLUENZA®, Hipra (Amer, Spain), in order to detect anti-SIV antibodies. In total, 24.13 % of samples from intensive herds were positive, while no positive samples were detected in extensive rearing herds. Sow and weaning piglets had the highest prevalence values. In the intensive rearing system, occurrence of reproductive disorders and exposure to recently introduced animals were positively associated with the disease occurrence in swine herds. The findings highlight the importance of sows in the epidemiology of the disease and bring information about risk factors involved in the occurrence of swine influenza in intensive herds.     

Loss of naïve (CD45RA+) CD4+ lymphocytes during pediatric infection with feline immunodeficiency virus

Feline immunodeficiency virus (FIV) infection of cats is an animal model for the pathogenesis of CD4+ lymphopenia and thymus dysfunction in HIV-infected humans. Recently, a monoclonal antibody (755) was reported to recognize the feline homologue to CD45RA, allowing the enumeration of na?ve T cells in cats. We tested the hypothesis that pediatric FIV infection would be associated with a selective loss of na?ve CD4+ lymphocytes by inoculating newborn cats with a pathogenic clone of FIV (JSY3) or a related clone with an inactive ORF-A gene (JSY3-DeltaORFA), and compared the data to age-matched uninfected control cats. Both FIV inocula were associated with a reduction in the CD4-CD8 ratio (p=0.01), which was attributable to a disproportionate loss of na?ve CD4+ cells (p=0.01) vs. na?ve CD8+ cells. Therefore, the reduced CD4:CD8 ratio in FIV-infected juvenile cats is associated with a selective depletion of na?ve CD4+ cells from the blood.     

Flow Cytometric Characterization and Clinical Outcome of CD4+ T‐Cell Lymphoma in Dogs: 67 Cases

Background

Canine T‐cell lymphoma (TCL) is conventionally considered an aggressive disease, but some forms are histologically and clinically indolent. CD4 TCL is reported to be the most common subtype of TCL. We assessed flow cytometric characteristics, histologic features when available, and clinical outcomes of CD4+ TCL to determine if flow cytometry can be used to subclassify this group of lymphomas.

Objective

To test the hypothesis that canine CD4+ T‐cell lymphoma (TCL) is a homogeneous group of lymphomas with an aggressive clinical course.

Animals

Sixty‐seven dogs diagnosed with CD4+ TCL by flow cytometry and treated at 1 of 3 oncology referral clinics.

Methods

Retrospective multivariable analysis of outcome in canine CD4+ TCL including patient characteristics, treatment, and flow cytometric features.

Results

The majority of CD4+ TCL were CD45+, expressed low class II MHC, and exhibited an aggressive clinical course independent of treatment regimen (median survival, 159 days). Histologically, CD4+ TCL were classified as lymphoblastic or peripheral T cell. Size of the neoplastic lymphocytes had a modest effect on both PFI and survival in this group. A small number of CD4+ TCL were CD45− and class II MHC high, and exhibited an apparently more indolent clinical course (median survival not yet reached).

Conclusions and Clinical Importance

Although the majority of CD4+ TCL in dogs had uniform clinical and flow cytometric features and an aggressive clinical course, a subset had a unique immunophenotype that predicts significantly longer survival. This finding strengthens the utility of flow cytometry to aid in the stratification of canine lymphoma.     

Prevalence of avian influenza,Newcastle disease,and infectious bronchitis viruses in broiler flocks infected with multifactorial respiratory diseases in Iran, 2015–2016

In this study, the prevalence and spatial distribution of Newcastle disease, infectious bronchitis, and avian influenza have been evaluated in commercial broiler farms in 31 provinces in Iran. In this survey, a total of 233 affected broiler chicken farms were sampled. The infectious bronchitis virus (alone) was detected with highest frequency in 60 farms, and separately or combined with other agents, in 110 farms; Newcastle disease virus, separately, was detected in 28 farms, and in 63 farms separately or combined with other infectious agents; and avian influenza H9N2 was detected in 22 farms separately and in 51 farms separately or concomitant with other infectious agents. The sample tested negative for all H5 serotypes. The results of the present study show that the most prevalent avian viral infectious disease contributing to respiratory syndromes in broiler farms in Iran was infectious bronchitis due to infectious bronchitis virus serotypes variant 2 and 793/B. On the other hand, combined with the alternation of dominant viruses and circulating strains, flocks are exposed to unremitting anamorphic viral infections. Thus, the permanent monitoring of cases that have occurred and the review of vaccination plans of affected flocks every year are some of the necessary measures needed for strategic control of respiratory syndrome in broilers. It is noteworthy that execution of epidemiologic examinations on the cogent factors of prevalence of this syndrome and defeat of vaccination strategy in the flocks is urgent and has to be fulfilled on the definite causes of time.

     

Sero‐epidemiological screening of pig sera collected at the slaughterhouse to detect herds infected with Aujeszky's disease virus,porcine influenza virus and Actinobacillus (Haemophilus) pleuropneumoniae in the framework of an Integrated Quality Control (IQC) system

Summary

Over a period of six months, approximately 4700 blood samples were collected from 97 pig‐finishing farms in the provinces of Noord‐Brabant and Gelderland and screened for antibodies with respect to Aujeszky's disease virus (ADV), porcine influenza virus (PI) and Actinobacillus (Haemophilus) pleuropneumoniae (App). There were significant differences in the percentages of seropositive pigs between the two provinces, which may be related to the difference in the density of the pig population in the two provinces.

In practice, it was possible to perform a reliable sera collecting procedure at the slaughterhouse. No farms remained seronegative with respect to most of the disease agents during the sampling period. There was a high degree of variation in the percentages of seropositive pigs per farm as to most of the disease agents. Evidence was found that animals that were seropositive with respect to ADV were significantly more susceptible to becoming seropositive with respect to App. serotype 2, and vice versa. The same connection was observed for PI serotype H₁N₁ and PI serotype H₃N₂. Furthermore, evidence was found that pigs seropositive with respect to PI serotype H₁N_i only, or to PI serotype H₁N₁ and ADV or PI serotype H₃N₂ show a significant decrease in average daily weight gain compared to pigs that were seronegative.     

Analysis of the situation of swine influenza in the districts Upper Bavaria, Swabia, Freiburg and Tübingen based on antibody profiles of piglets from Bavarian breeding farms

A total of 1026 serum samples from 388 pigs from three Bavarian rearing farms in the region of Swabia were investigated in the course of investigations into the development of antibodies against Influenza A virus subtypes H1N1, H1N2 and H3N2 by haemagglutination inhibition test during the period from November 2002 to February 2004. There were no signs of respiratory disease during this period. The antibody titres decreased steadily in this period which corresponds to the kinetics of maternally-derived antibodies. Therefore the antibodies reflect the situation of influenza in the farms of origin from which the piglets were purchased. These farms were located in Bavaria and Baden-Wuerttemberg. There was a high activity of H1N1 influenza A viruses in this region whereas the antibody profiles against H1N2 and H3N2 varied between the different farms, which can be attributed to past vaccinations and infections. Thus there was a uniform immunological situation within the regions against H1N1 whereas that against H1N2 and H3N2 differed. The analysis of the antibody profiles allows conclusions to be drawn about the epidemiological situation and means of immunoprophylaxis.     

Effects of parturition and dexamethasone on DNA methylation patterns of IFN-γ and IL-4 promoters in CD4+ T-lymphocytes of Holstein dairy cows

This study investigated epigenetic mechanisms by which DNA methylation affects the function of bovine adaptive immune system cells, particularly during the peripartum period, when shifts in type 1 and type 2 immune response (IR) biases are thought to occur. Stimulation of CD4+ T-lymphocytes isolated from 5 Holstein dairy cows before and after parturition with concanavalin A (ConA) and stimulation of CD4+ T-lymphocytes isolated from 3 Holstein dairy cows in mid-lactation with ConA alone or ConA plus dexamethasone (Dex) had significant effects on production of the cytokines interferon gamma (IFN-γ, type 1) and interleukin 4 (IL-4, type 2) that were consistent with DNA methylation profiles of the IFN-γ gene promoter region but not consistent for the IL-4 promoter region. ConA stimulation increased the production of both cytokines before and after parturition. It decreased DNA methylation in the IFN-γ promoter region but increased for IL-4 promoter region. Parturition was associated with an increase in IFN-γ production in ConA-stimulated cells that approached significance. Overall, DNA methylation in both promoter regions increased between the prepartum and postpartum periods, although this did not correlate with secreted cytokine concentrations. Dexamethasone treated cells acted in a manner consistent with the glucocorticoid’s immunosuppressive activity, which mimicked the change at the IFN-γ promoter region observed during parturition. These results support pregnancy as type 2 IR biased, with increases of IFN-γ occurring after parturition and an increase in IL-4 production before calving. It is likely that these changes may be epigenetically controlled.     

Do animal exhibitors support and follow recommendations to prevent transmission of variant influenza at agricultural fairs? A survey of animal exhibitor households after a variant influenza virus outbreak in Michigan

Influenza A viruses circulate in swine and can spread rapidly among swine when housed in close proximity, such as at agricultural fairs. Youth who have close and prolonged contact with influenza‐infected swine at agricultural fairs may be at increased risk of acquiring influenza virus infection from swine. Animal and human health officials have issued written measures to minimize influenza transmission at agricultural exhibitions; however, there is little information on the knowledge, attitudes, and practice (KAP) of these measures among animal exhibitors. After an August 2016 outbreak of influenza A(H3N2) variant (“H3N2v”) virus infections (i.e., humans infected with swine influenza viruses) in Michigan, we surveyed households of animal exhibitors at eight fairs (including one with known H3N2v infections) to assess their KAP related to variant virus infections and their support for prevention measures. Among 170 households interviewed, most (90%, 151/167) perceived their risk of acquiring influenza from swine to be low or very low. Animal exhibitor households reported high levels of behaviours that put them at increased risk of variant influenza virus infections, including eating or drinking in swine barns (43%, 66/154) and hugging, kissing or snuggling with swine at agricultural fairs (31%, 48/157). Among several recommendations, including limiting the duration of swine exhibits and restricting eating and drinking in the animal barns, the only recommendation supported by a majority of households was the presence of prominent hand‐washing stations with a person to monitor hand‐washing behaviour (76%, 129/170). This is a unique study of KAP among animal exhibitors and highlights that animal exhibitor households engage in behaviours that could increase their risk of variant virus infections and have low support for currently recommended measures to minimize infection transmission. Further efforts are needed to understand the lack of support for recommended measures and to encourage healthy behaviours at fairs.     

Treatment of cattle with DNA-encoded Flt3L and GM-CSF prior to immunization with Theileria parva candidate vaccine antigens induces CD4 and CD8 T cell IFN-γ responses but not CTL responses

Theileria parva antigens recognized by cytotoxic T lymphocytes (CTLs) are prime vaccine candidates against East Coast fever in cattle. A strategy for enhancing induction of parasite-specific T cell responses by increasing recruitment and activation of dendritic cells (DCs) at the immunization site by administration of bovine Flt3L and GM-CSF prior to inoculation with DNA vaccine constructs and MVA boost was evaluated. Analysis of immune responses showed induction of significant T. parva-specific proliferation, and IFN-γ-secreting CD4(+) and CD8(+) T cell responses in immunized cattle. However, antigen-specific CTLs were not detected. Following lethal challenge, 5/12 immunized cattle survived by day 21, whereas all the negative controls had to be euthanized due to severe disease, indicating a protective effect of the vaccine (p<0.05). The study demonstrated the potential of this technology to elicit significant MHC class II and class I restricted IFN-γ-secreting CD4(+) and CD8(+) T cells to defined vaccine candidate antigens in a natural host, but also underscores the need to improve strategies for eliciting protective CTL responses.