Systemic, mucosal, and heterotypic immune induction in mice inoculated with Venezuelan equine encephalitis replicons expressing Norwalk virus-like particles

Abstract

Norwalk-like viruses (NLVs) are a diverse group of single-stranded, nonenveloped, positive-polarity RNA viruses and are the leading cause of epidemic acute gastroenteritis in the United States. In this study, the major capsid gene of Norwalk virus, the prototype NLV, has been cloned and expressed in mammalian cells using a Venezuelan equine encephalitis (VEE) replicon expression system. Upon infection of baby hamster kidney (BHK) cells with VEE replicon particles (VRPs), the Norwalk virus capsid proteins self-assemble to generate high titers of Norwalk virus-like particles (VLPs) that are morphologically and antigenically analogous to wild-type Norwalk virus. Mice inoculated subcutaneously with VRPs expressing the Norwalk virus capsid protein (VRP-NV1) developed systemic and mucosal immune responses to Norwalk VLPs, as well as heterotypic antibody responses to the major capsid protein from another genogroup I NLV strain (NCFL) isolated from a recent outbreak. A second Norwalk virus capsid clone (NV2) containing three amino acid codon mutations from the NV1 clone was also expressed using VEE replicons (VRP-NV2), but upon infection of BHK cells failed to confer VLP self-assembly. Mice inoculated with VRP-NV2 elicited reduced systemic and mucosal immune responses to Norwalk VLPs, demonstrating the importance and potential utility of endogenous VLP presentation for maximum immune induction. Inoculation with either VRP-NV1 or VRP-NV2 resulted in serum antibody responses far superior to the induction in mice dosed orally with VLPs that were prepared using the VEE-NV1 replicon construct, a regimen similar to current models for NLV vaccination. Expression of NLV VLPs in mammalian cells offers a powerful approach for the design of novel NLV vaccines, either alone or in combination with current vaccination models.

FIG. 1.

NLV capsid clones. NV1, NV2, and NCFL NLV capsid genes were cloned from fecal specimens and sequenced. NV1 and NV2 were isolated from human volunteers infected with the NV8FIIa challenge inoculum (Moe et al., unpublished data), and NCFL was isolated from an infected patient from a recent NLV outbreak that occurred during a football game in North Carolina (4). Amino acid variations and identities to Chiba virus and Norwalk virus capsid (37, 62) are indicated in panel a. Panel b shows an amino acid alignment of NV1, NCFL, and Chiba capsids. Differences from the Chiba reference are indicated in boldface. Note that the designated amino acid numbers in panel b might be shifted due to the alignment.

FIG. 1.

NLV capsid clones. NV1, NV2, and NCFL NLV capsid genes were cloned from fecal specimens and sequenced. NV1 and NV2 were isolated from human volunteers infected with the NV8FIIa challenge inoculum (Moe et al., unpublished data), and NCFL was isolated from an infected patient from a recent NLV outbreak that occurred during a football game in North Carolina (4). Amino acid variations and identities to Chiba virus and Norwalk virus capsid (37, 62) are indicated in panel a. Panel b shows an amino acid alignment of NV1, NCFL, and Chiba capsids. Differences from the Chiba reference are indicated in boldface. Note that the designated amino acid numbers in panel b might be shifted due to the alignment.

FIG. 2.

Expression and self-assembly of NLV capsid proteins. BHK cells were either infected with VEE VRPs expressing NV1 or NV2, mock infected, or transfected with VEE replicon RNA expressing the NCFL capsid gene. (A) IFA for NV1, NV2, and NCFL capsid protein expression. Cells were examined in a fluorescent microscope for NLV capsid expression using human antiserum to either Norwalk virus (NV1, NV2, and Mock) or the NCFL isolate (NCFL) and goat anti-human IgG-FITC-conjugated secondary antibody. (B) BHK cell lysates were examined for evidence of NLV VLP self-assembly. Putative VLPs were collected by ultracentrifugation through sucrose gradients and analyzed by negative-stain electron microscopy. Scale bar, 100 nm. (C) NV1 and NV2 capsid proteins from VRP-infected BHK cell lysates were analyzed by Western blot using human antiserum to Norwalk virus. Lanes marked 1x and 2x represent uninfected BHK cell lysates at 1 and 2 times the total loaded protein, respectively, versus the NV1 and NV2 lanes. Arrows indicate Norwalk virus antiserum-reactive protein bands, and arrowheads indicate background bands.

FIG. 3.

Serum immune responses to Norwalk VLPs (VEE-Nor). Groups of four mice were inoculated on days 0 and 23 (indicated by arrows in IgG chart) according to Table 1. Serum samples were collected and analyzed by ELISA for VEE-Nor-specific serum IgM or IgG. Experimental regimens and sample time points are indicated. Error bars at each time point represent the standard deviation between mice. *, three of four mice represented; one mouse developed IgM levels nearly 10 times the average of the other three mice at this time point (data not shown). Data from this mouse are included for all remaining time points.

FIG. 4.

Intestinal IgA responses to VEE-Nor. Fecal pellets were collected from all mice in each mouse group at the indicated time points, processed to obtain a clarified, buffered extract, and analyzed by ELISA. Samples are from the same mice inoculated on days 0 and 23 represented in Table 1 and Fig. 3. To correct for background, IgA values for each time point from mock-infected mice were subtracted from corresponding time points for all experimental groups.

FIG. 5.

Serum IgG boost responses from NV1:VEE-Nor mice. Average serum IgG titers of four mice on days 14, 21, and 28, which were inoculated on day 0 with VRP-NV1 and boosted orally on day 23 (arrow) with 75 μg of VEE-Nor. Error bars represent standard deviation. *, the increase in IgG titers from day 14 to day 21 was not statistically significant (P > 0.1); **, significant increase in IgG titers from day 21 to day 28 (P < 0.01; Student’s paired t test of data from two separate ELISA analyses).

FIG. 6.

Heterotypic immune responses from VRP-NV1-inoculated mice. BHK cells were electroporated with NCFL-encoding VEE replicon RNA, and cross-reactive IgG responses were examined by IFA. Reactivity was detected using (A) serum from an NCFL-infected patient, (B) serum from a Norwalk virus-infected volunteer, (C) pooled high-titer serum from VRP-NV1-inoculated mice, and (D) preimmune mouse serum, followed with goat anti-human or goat anti-mouse IgG-FITC-conjugated secondary antibodies.