Summary A randomised, controlled, double‐blind, influenza virus, aerosol challenge of horses was undertaken to determine the efficacy of a cold‐adapted, temperature sensitive, modified‐live virus, intranasal, equine influenza vaccine. Ninety 11‐month‐old influenza‐naïve foals were assigned randomly to 3 groups (20 vaccinates and 10 controls per group) and challenged 5 weeks, 6 and 12 months after a single vaccination. Challenges were performed on Day 0 in a plastic‐lined chamber. Between Days 1 and 10, animals were examined daily for evidence of clinical signs of influenza. Nasal swabs for virus isolation were obtained on Day 1 and Days 1 to 8 and blood samples for serology were collected on Days 1, 7 and 14. There was no adverse response to vaccination in any animal. Following challenge at 5 weeks and 6 months, vaccinates had significantly lower clinical scores (P = 0.0001 and 0.005, respectively), experienced smaller increases in rectal temperature (P = 0.0008 and 0.0007, respectively) and shed less virus (P<0.0001 and P = 0.03, respectively) over fewer days (P<0.0001 and P = 0.002, respectively) than did the controls. After the 12 month challenge, rectal temperatures (P = 0.006) as well as the duration (P = 0.03) and concentration of virus shed (P = 0.04) were significantly reduced among vaccinated animals. The results of this study showed that 6 months after a single dose of vaccine the duration and severity of clinical signs were markedly reduced amongst vaccinated animals exposed to a severe live‐virus challenge. Appropriate use of this vaccine should lead to a marked reduction in the frequency, severity and duration of outbreaks of equine influenza in North America.

Summary Flu Avert IN vaccine is a new, live attenuated virus vaccine for equine influenza. We tested this vaccine in vivo to ascertain 1) its safety and stability when subjected to serial horse to horse passage, 2) whether it spread spontaneously from horse to horse and 3) its ability to protect against heterologous equine influenza challenge viruses of epidemiological relevance. For the stability study, the vaccine was administered to 5 ponies. Nasal swabs were collected and pooled fluids administered directly to 4 successive groups of naïve ponies by intranasal inoculation. Viruses isolated from the last group retained the vaccine's full attenuation phenotype, with no reversion to the wild‐type virus phenotype or production of clinical influenza disease. The vaccine virus spread spontaneously to only 1 of 13 nonvaccinated horses/ponies when these were comingled with 39 vaccinates in the same field. For the heterologous protection study, a challenge model system was utilised in which vaccinated or naïve control horses and ponies were exposed to the challenge virus by inhalation of virus‐containing aerosols. Challenge viruses included influenza A/equine‐2/Kentucky/98, a recent representative of the ‘American’ lineage of equine‐2 influenza viruses; and A/equine‐2/Saskatoon/90, representative of the ‘Eurasian’ lineage. Clinical signs among challenged animals were recorded daily using a standardised scoring protocol. With both challenge viruses, control animals reliably contracted clinical signs of influenza, whereas vaccinated animals were reliably protected from clinical disease. These results demonstrate that Flu Avert IN vaccine is safe and phenotypically stable, has low spontaneous transmissibility and is effective in protecting horses against challenge viruses representative of those in circulation worldwide.

SununaryAntibodies bearing the T15 idiotype dominate the murine primary immune response to phosphocholine (PC). Analysis of antigen binding of antibodies derived from VI:DFL16.1:J.1 (V.1) germline and N region-derived variant heavy (H) chains and g22, K24, and K8 light (L) chains demonstrates that the T15H:K22L (T15) antibody binds PC at least 20-40 times better than other antibodies derived from alternate germline forms of the V.1 H chain and g22, K24, or K8 L chains. To achieve af~nities in the same range as the T15 antibody, K24 and Ic8 L chain-containing antibodies must have H chains derived from variant N region or somatically mutated V.1 genes. Single amino acid differences at the VD junction of the various germline and N region variant V.1 H chains dictate the L chain that can associate with the H chain to produce a PC-specific antibody. Several H:L combinations give rise to T15 or M167 idiotypepositive antibodies that lack specificity for PC, and single amino acid substitutions or insertions at the V.I:D junction result in the loss of T15 or M167 idiotopes. Based on these observations, our data support a molecular model involving both preferential gene rearrangement and antigendriven B cell selection to explain T15 idiotype dominance in the immune response to PC. In the absence of N region diversification, large numbers of neonatal B cells bearing the T15H:K22L surface immunoglobulin M (slgM) receptors would be selected and expanded by autologous or environmental PC antigen into the long-lived peripheral B cell pool.T he primary antibody response to PC in normal inbred mice is dominated by antibodies expressing H and L chain products of the V.1 and V~22 germline genes (1, 2). These antibodies are identical to the antibody produced by the T15 plasmacytoma (3). The T15 clone of B cells is present in the mouse spleen at a higher frequency than any other individual clone of B cells and comprises >75% of all phosphocholine (PC)l-specific B cells in normal mice (4). The reasons for T15-Id dominance of the anti-PC immune response are not fully understood; however, idiotype network selection, binding-site affinity, and preferential V gene expression have I Abbreviation used in this paper: PC, phosphocholine. all been suggested. A. J. Feeney (5) has recently shown that over 50% of the V1:DFL16.1junctions produced in neonatal pre-B cells have prototypic T15 V-D junctional sequences, and has hypothesized that this preferential recombination of these germline genes would account for the dominance of T15 clones in the anti-PC repertoire. If the prototypic T15H chain is preferentially produced and this H chain can only form a PC-specific antibody with a K22 L chain, the subsequent receptor-driven selection and amplification of this clone into a long-lived B cell pool could account for T15-Id dominance. This hypothesis is supported by our recent observation (6, 7) that PC-specific B cells in M167 H chain transgenic mice are selected and amplified by a receptor-mediated, antigen-driven process after they emerge from ...

We have initiated a series of experiments to explore the effect of changes in density of the surface Ig receptor (mIg) on Ag:mIg interactions. We transfected into the surface Ig-negative B cell line M12.4 H and L chain constructs known to effect a 10-fold change in antibody binding affinity for the naturally occurring hapten phosphocholine (PC). Two sets of stable transfectants were generated and those expressing levels of mIg comparable to the range normally seen on splenic B cells were studied. One set expressed an unmutated VH and an unmutated VL. The second set expressed the same pair of V regions except for a single somatic change in CDR3 of VH; this substitution increases the affinity of antibody for PC from 3 x 10(4) M-1 to 3 x 10(5) M-1. Ag:mIg interactions were assessed in the transfected cell lines by measuring calcium mobilization induced by stimulation with soluble PC Ag. As expected, the mutation that increased affinity for PC increased the sensitivity of transfectants to PC Ag. Relatively small changes in receptor number had a dramatic effect in the quantity and quality of a calcium response. Significantly, we found that Ag-specific signaling could occur with only a few thousand receptors per cell. Signaling differences were most noticeable with PC protein Ag (T-dependent form) compared with PC polysaccharide Ag (T-independent form). These results suggest that the down-regulation of mIg that follows B cell activation may have evolved to assist in the selection of B cell clones with higher affinity for Ag. Furthermore, the results also provide an explanation for why selection of higher affinity clones can occur with protein Ag but only poorly so with polymeric Ag.

We wished to resolve a paradox of how the response to the phosphocholine (PC) determinant of Proteus morganii could be initiated from a precursor B cell whose receptor, in unmutated form as Ab, appears to be unable to bind Ag. Unmutated VH and unmutated VL constructs were co-transfected into the B cell lymphoma M12.4 to study stimulation via membrane lg (mlg). The same VH construct was expressed in an L+, H- hybridoma line to characterize Ab binding. The unmutated Ab showed no detectable binding in ELISA to the PC-containing Ag from P. morganii PC(PM). By contrast, the unmutated mlg mediated mobilization of calcium in response to the PC(PM) Ag. Single-positive B cell lines of mlgM, mlgD double-positive lines were all capable of responding. The degree of signaling depended greatly on high receptor number, and only a fraction of cells in the population responded. Inhibition of the PC(PM)-induced calcium response by free PC indicated that the response was Ag-specific. A transfectant B cell line expressing moderate levels of a high affinity, mutated mlgM readily responded to PC(PM). These observations indicate that the unmutated lg as a receptor is capable of interacting with PC(PM) and suggest that the immune response to PC(PM) could originate from the precursor B cell expressing the unmutated mlg. The role of mlgD vs mlgM is discussed in terms of the requirement for high receptor number in the signaling process.