Dissecting Immune Responses. Miriam Windsor, Nicholas Juleff, Mandy Corteyn, Pippa Hamblin, Veronica Carr, Paul V Barnett, Bryan Charleston*

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1 Appendix 28 Dissecting Immune Responses Miriam Windsor, Nicholas Juleff, Mandy Corteyn, Pippa Hamblin, Veronica Carr, Paul V Barnett, Bryan Charleston* Abstract: Pirbright Laboratory, Institute for Animal Health, Ash Rd, Woking, Surrey, GU24 NF, UK. Objectives: To understand the role of CD4 T cells in protective immune responses against FMDV infection of cattle. The role of CD4 T cells was investigated during acute infection of naïve animals and the magnitude of specific CD4 responses was correlated with protection in vaccinated animals. Materials and methods: Two separate animal studies have been carried out. In the first experiment naïve animals were depleted of circulating CD4 T cells with monoclonal antibodies before intradermolingual inoculation with the O UKG 34/21 strain of FMDV. Neutralizing antibody titres, clinical signs and memory CD4 T cell responses were measured pre and post challenge. In the second series of experiments, animals were vaccinated with O serotype vaccine from the UK emergency vaccine bank. Total and neutralizing anti-fmdv antibody titres were measured at time points post-vaccination and these titres correlated with the magnitude of the specific T cell response. One year post vaccination one group of animals was challenged intradermolingually with homologous virus. Results: Elimination of CD4 T cells using monoclonal antibody depletion did not influence the magnitude or duration of clinical signs or reduce the development of neutralizing antibody titres post-challenge. There was a correlation between the duration and magnitude of the humoral response with the magnitude of the cellular immune response. Also, assessing the cellular immune response improved the ability to predict protection after intradermolingual challenge with homologous virus. Discussion: Our preliminary results suggest that the early antibody production in naïve cattle to FMDV is predominantly a T independent response. Also, the development and control of clinical signs are not dependent on the presence of circulating CD4 T cells. Using neutralizing antibody titre combined with specific CD4 T cell proliferative response could provide an improved correlation with protection from FMDV clinical signs, compared to antibody titre alone. Introduction: Foot and mouth disease virus (FMDV) has a wide host range including all cloven-hoofed animals and causes an acute vesicular disease in domestic ruminants and pigs, which results in debilitation, pain and loss of productivity (Alexandersen et al., 23). Commercial vaccines, which comprise inactivated virus particles incorporated in adjuvant, are widely used in those parts of the world where FMD is prevalent (Barnett and Carabin 22, Barnett et al. 21). However, despite its widespread use, the vaccine has a number of shortcomings (Doel 1999). Immunity is relatively short-lived and hence animals need to be re-vaccinated at 4-6 month intervals. By contrast available data, although limited, suggest that immunity in animals that have recovered from infection with FMDV may last up to 3 years (Cuncliffe 1964). Novel approaches to the development of new vaccines against FMD are limited by a relatively superficial understanding of the immunology of the disease in the target species. Evidence from model virus systems in laboratory animals has shown that, while either antibody or T cell-mediated effector mechanisms can have a dominant role in immunity to different viruses, both arms of the immune response are often required to maximise protection antibody to neutralize and remove free virus, and T cells to inhibit viral replication or to kill-virus-infected cells (Zinkernagel 22). The CD4 and CD8 subsets of T cells both have the potential to act as effectors; the former are also generally required to provide help for antibody production. In the context of FMDV, there are a number of differences between the immune responses induced by infection and vaccination that potentially could influence the quality and/or duration of immunity to FMDV. 188

2 By contrast to antibody, knowledge of T cell responses to FMDV is limited. CD4 T cell responses have been described in infected and vaccinated animals and in both cases were found to be crossreactive between virus serotypes (Collen et al, 1998). CD4 T cells from infected animals recognised both structural and non-structural proteins. Intriguingly, the dominant viral protein recognized by vaccinated animals was 3D (Collen et al, 1998), which is a non-structural protein but recently has been identified as a minor component of intact virions (Newman et al., 1994). Vaccinated animals also respond to structural proteins and several CD4 T cell epitopes have been identified in these proteins (Collen et al, 1991; van Lierop et al, 1994 and 1995). One study of vaccinated animals reported that the responding CD4 T cells express IL-2 and IFN-γ but not IL-4, suggesting a Th1- type response (van Lierop et al, 1995). Experiments in T cell-deficient mice have provided evidence that, in common with a number of other viruses, FMDV can induce T-cell-independent antibody responses (Borca et al, 1986). Evidence from other viral systems indicates that this is a consequence of the repetitive arrangement of B cell epitopes on the surface of the viral particles. Bachmann and Zinkernagel (1996) have argued that viruses with this property are able to generate very rapid antibody responses because early expansion of B cells by T cell-independent mechanisms facilitates an accelerated T-dependent antibody response. If this is correct, it has important implications for vaccine design since induction of this response would require retention of a complex antigen configuration in the vaccine. Studies have been initiated to determine the T cell dependency of antibody response to FMDV in cattle and to investigate the extent to which immunity in vaccinated animals is dependent on a strong CD4 T cell memory response. Materials and Methods: Animals: The animals used in the studies to correlate antibody response with T cell proliferative response were derived from a herd under a controlled breeding scheme where a selection according to MHC haplotypes is applied. The animals used showed serological specificity for either BoLA class II allele DRB3*71or DRB3*22 (Collen et al., 22). Specific T cell subset depletion studies were performed in commercial out-bred calves. Clinical scoring: A clinical scoring system based on rectal temperature, the occurrence of lesions on feet and mouth, lameness and nasal discharge was used. Monoclonal antibody depletion of T cell subsets: Calves weighing approximately 8kg were treated over a three day period with 5mg of purified IgG2a monoclonal antibodies against either CD4, WC1 antigen (expressed on circulating gamma delta T cells) or control antibody (specific for Turkey Rhinotracheitis virus). Monoclonal antibody depletion was initiated one day prior to challenge. Lymphocyte proliferation assays: Heparinized blood was collected and peripheral blood mononuclear cells (PBMC) isolated according to a standard procedure. PBMC were resuspended in RPMI 164 with L-glutamine (Gibco), supplemented with 1 % fetal calf serum (Biochrom, UK) and plated (2 x 1 5 cells/well) in 96-well round-bottomed microtitre plates (Falcon). Control and test antigens were added to each well at concentrations described in the results. After five days, cultures were pulsed with 1 µci of methyl- 3 H-thymidine for 18 hours. Cells were collected and incorporation of 3 H-thymidine was measured by liquid scintillation counting. Flow cytometry: All stages were performed at room temperature. Cells were seeded at 2 x 1 5 cells/well in a u-bottomed 96 well plate. The cells were washed in FACS Wash Buffer (FWB) by centrifugation at 13rpm for 3 minutes. The supernatant was flicked off and the cells vortexed to resuspend them. Primary antibodies were added at 1µg/ml in a total volume of 25µl. The cells were incubated for 1 minutes followed by two washes. The cells were then incubated with 25µl of isotype specific fluorochrome for 1 mins, protected from light, followed by another two washes. The cells were then diluted into 35µl FWB and analyzed immediately on a FACS machine. If analysis could not be performed immediately, cells were fixed with 1µl 1% paraformaldehyde, stored at 2-8 C and protected from light. Recovery of cells was performed by adding 1µl FWB to wells and centrifuging as above. Live cells were gated according to their forward and side scatter profiles. Results were analyzed on FACSExpress V3. CD4 proliferation assays: PBMC were isolated from whole cow blood by density gradient centrifugation. Blood was diluted 1:1 with D-PBS, underlaid with Histopaque-183 and centrifuged for 3 minutes at 25rpm at room temperature. Subsequent centrifugations were carried out at 8 C. PBMCs were removed from the interface and washed in D-PBS by centrifugation, once at 18rpm for 1 minutes and twice at 12rpm for 8 minutes. Prior to the third wash, a 5µl sample of cells were diluted in 95µl of.1% trypan blue for counting. 189

3 An aliquot of PBMC were isolated irradiated and seeded into u-bottomed 96 well plates at 3 x 1 3 cells/well in 1µl of media. PBMCs were initially incubated with 5µl of cc3 (IgG1 antibody) in 5mls of D-PBS for 1 minutes at room temperature. The suspension was then washed twice in D- PBS and the cell pellet incubated with anti-igg1 magnetic beads for 1 minutes at room temperature. After two more washes, cells were positively selected on an LS MidiMACS column. Cells were counted and seeded over the antigen-presenting cells at a concentration of 1x 1 5 cells / well in a volume of 5µl. Antigens were added in triplicate at the appropriate concentrations in a total volume of 1µl. Pokeweed mitogen was used as a positive control and background was assessed in wells containing only antigen presenting cells and CD4+ cells. FMD virus neutralization test: Titres of neutralizing FMD antibodies were measured by the microneutralization assay as described in the OIE Manual in which antibody end point titres are calculated as the reciprocal of the last serum dilution to neutralize 1 TCID 5 of the virus per well. The same strain of virus was used in the test as in the in vivo cattle potency test. 19

4 Results: In vitro antigen specific proliferation of peripheral blood mononuclear cells to FMDV antigen is dependent on the presence of CD4 T cells. Two animals with established specific proliferative responses to FMDV antigen post vaccination were selected to determine whether the specific proliferative response was dependent on CD4 T cells. The results in Figure 1 show the animals have a specific response to FMDV (a). Depletion of CD 4 T cells from the PBMC results in a loss of a specific proliferative response (b). However, the addition of purified CD4 cells to irradiated whole PBMC as antigen presenting cells results in a detectable specific proliferative response (c). FMD 2 PBMC responses FMD 4 PBMC responses a 8 6 Mean cpm 4 FMD 2 PBMC-CD4 responses Antigen FMD 4 PBMC-CD4 responses b 8 6 Mean cpm 4 Antigen FMD 2 CD4 +APC responses FMD 4 CD4 + APC responses c 8 Mean cpm 6 4 Media PWM Ova Cells only Media PWM Ova Cells only Antigen Figure 1 191

5 The magnitude of the FMDV specific T cell proliferative response is associated with neutralizing antibody titre and protection. a 14 c VNT FMD 1 FMD 2 FMD 3 FMD 4 FMD 5 Virus Neutralisation titre FMD6 FMD7 FMD8 FMD9 FMD1 Days , 2 and 4 re-immunised Weeks post Vaccination b 6 d Figure 2 Initial observations relating antibody titre to specific proliferative response of total PBMC of cattle vaccinated with O Manisa commercial vaccine are shown in Figure 2. (a) Neutralizing antibody response of five vaccinated cattle assessed for 1 year post-vaccination (MHCII haplotype DRB3* 71). All animals had a neutralizing antibody titre of 1/45 or less. (b) Specific proliferative response to FMDV (open bars) and Ovalbumin (closed bars) on the day of challenge (1 Year post first vaccination). Only animals 2 and 4 were protected from homologous challenge. (c) Specific virus neutralizing antibody response of five animals (MHCII haplotype DRB3* 22) immunized with commercial O Manisa vaccine. All animals were re-vaccinated after 1 weeks and the specific virus neutralizing antibody response measured 3 weeks later. (d) The specific proliferative response to FMDV (open bars) and Bovine Herpes Virus (closed bars) 3 weeks post second vaccination is shown. There is a tendency for animals with high antibody titre to also have a higher specific proliferative response. 192

6 Depletion of CD4 T cells or WC1+ γδ T cells does not influence the development and resolution of clinical signs or circulating neutralizing antibody. After challenge of the monoclonal antibody treated animals with viral inoculum the onset and duration of clinical signs was similar in all animals. One of the CD4 T cell depleted animals showed less severe lesions, although the onset and duration of those lesions were consistent with the other calves. All calves were clinically recovered by 7 days post-challenge except for the presence of healing lesions (Figure 3). The effectiveness of the monoclonal antibody depletion protocol was assessed by determining the percentage of the target cell population in circulation at various timepoints pre and post-treatment. The target populations were effectively depleted for at least 7 days (Figure 4 d-f). All animals had neutralizing antibody titres greater than 1/45 by day 7 postchallenge (Figure 4 a-c) Figure 3 Clinical scores post-fmdv challenge of calves depleted of either CD4 or WC1+ γδ T cells. Control animals ; CD4 T cell depleted animals ; γδ T cell depleted animals 193

7 a TRT3 Cont r ol s VNT RZ 57 ( Depletion control) d % lymphocytes WC1 CD4 CD8 CD Days post i nf ect i on b CD4 depleted VNT CD4 Depletion e VNT titre % lymphocytes c Gamma delta depleted VNT WC1 Depletion f 8 3 VNT titre % lym phocytes RZ 51 RZ Figure 4 Panels (a-c) neutralizing antibody titres post-challenge for control (a), CD4 depleted (b) and WC-1 depleted (c). Panel (d) shows the percentage of individual mononuclear cell populations in the peripheral blood animals treated with control antibody. Panel (e) and (f) shows the percentage of CD4 T cells and WC-1 T cells respectively, after monoclonal antibody treatment. 194

8 Discussion: Understanding the key elements of the immune response responsible for protection provides a focus for further vaccine development. However, the correlates of protection involved in resolution of infection do not necessarily reflect those that would be protective in the presence of pre-existing vaccine induced immunity. In the studies reported here, our preliminary results suggest the magnitude and duration of the neutralizing antibody response are related to the magnitude of the CD4 T cell response. Also, it would appear that measuring the specific T cell response to FMDV after vaccination may improve the ability to predict whether animals will be protected from subsequent challenge. Further development of the assays to measure specific CD4 T cell responses may improve the predictive capacity of these assays. The usefulness of measuring cell proliferation or interferon gamma production of whole PBMC populations is compromised because a significant component of the responses measured are due to bystander activation of other cell populations, for example gammadelta T cells. These difficulties can largely be resolved by analyzing purified cell populations; unfortunately cell sorting is still a relatively expensive and time-consuming process. Advances in the development of multiparameter flow cytometry may allow the simultaneous assessment of multiple cytokines produced from antigen specific CD4 T cells. Pre-existing antibody provides a mechanism for immediate protection to virus challenge. In the animals challenged one year after first vaccination, where protection was related to the magnitude of the T cell response rather than antibody titre, it is possible the presence of previously primed CD4 T cells may promote a more rapid and effective antibody response. In contrast, resolution of clinical signs after acute infection with FMDV appears to be independent of CD4 T cells, WC+ γδ T cells and detectable circulating antibody. These findings corroborate the ideas of Grubman (25), who have demonstrated the effectiveness of type-1 interferon in controlling FMDV infection. Conclusions: Further refinement of assays to measure specific T cell responses to FMDV should be performed alongside vaccine development programmes, to enhance our understanding of protective immune mechanisms and consequently influence vaccine design. References: Alexandersen S., Zhang., Donaldson A. I. & Garlean A. J. M. (23) The pathogenesis and diagnosis of foot-and-mouth disease. J. Comp. Path. 129, Barnett PV & Carabin H. (22) A review of emergency foot-and-mouth disease (FMD) vaccines. Vaccine, 2, Barnett PV, Samuel AR & Statham RJ. (21) The suitability of the 'emergency' foot-andmouth disease antigens held by the International Vaccine Bank within a global context. Vaccine, 19, Bachmann MF & Zinkernagel RM. (1997) Neutralizing antiviral B cell responses. Annu Rev Immunol, 15, Borca MV, Fernandez FM, Sadir AM, Braun M & Schudel AA. (1986) Immune response to foot-and-mouth disease virus in a murine experimental model: effective thymus-independent primary and secondary reaction. Immunology, 59, Collen T., Baron J., Childerstone A., Corteyn A., Doel T. R., Flint M., Garcia-Valcarcel M., Parkhouse R. M. E. & Ryan M. D. (1998) Heterotypic recognition of recombinant FMDV proteins by bovine T-cells: the polymerase (P3Dpol) as an immunodominant T-cell immunogen, Virus Research 56, Collen T., Carr V., Parsons K., Charleston B. & Morrison W. I. (22) Analysis of the repertoire of cattle CD4(+) T cells reactive with bovine viral diarrhoea virus, Vet. Immunol. Immunopathol. 87, Collen T., Dimarchi R.& Doel T. R. (1991) A T cell epitope in VP1 of foot-and-mouth disease virus is immunodominant for vaccinated cattle, J. Immunol. 146,

9 Cunliffe H. R. Observations on the duration of immunity in cattle after experimental infection with foot-and-mouth-disease-virus. Cornell Vet. 1964,54, Doel T. R. Optimisation of the immune response to foot-and-mouth disease vaccines. Vaccine. 1999, 17, Grubman M. (25) Development of novel strategies to control foot-and-mouth disease: marker vaccines and antivirals. Biologicals, 33: Newman JF, Piatti PG, Gorman BM, Burrage TG, Ryan MD, Flint M, Brown F. (1994) Footand-mouth disease virus particles contain replicase protein 3D. Proc Natl Acad Sci, 91, Van Lierop M. J., Arik T. J., van Noort J. M., Wagenaar J. P., Rutten V. P., Langeveld J., Meloen R. H. & Hensen E. J. (1994) T cell-stimulatory fragments of foot-and-mouth disease virus released by mild treatment with cathepsin D, J. Gen. Virol Van Lierop M. J., Wagenaar J. P., van Noort J. M. & Hensen E. J. (1995) Sequences derived from the highly antigenic VP1 region 14 to 16 of foot-and-mouth disease virus do not prime for a bovine T-cell response against intact virus, J. Virol Zinkernagel R. M. (22) On differences between immunity and immunological memory. Curr Opin Immunol., 14,