A nonproliferating parvovirus vaccine vector elicits sustained, protective humoral immunity following a single intravenous or intranasal inoculation

Abstract

An ideal vaccine delivery system would elicit persistent protection following a single administration, preferably by a noninvasive route, and be safe even in the face of immunosuppression, either inherited or acquired, of the recipient. We have exploited the unique life cycle of the autonomous parvoviruses to develop a nonproliferating vaccine platform that appears to both induce priming and continually boost a protective immune response following a single inoculation. A crippled parvovirus vector was constructed, based on a chimera between minute virus of mice (MVM) and LuIII, which expresses Borrelia burgdorferi outer surface protein A (OspA) instead of its coat protein. The vector was packaged into an MVM lymphotropic capsid and inoculated into naive C3H/HeNcr mice. Vaccination with a single vector dose, either intravenously or intranasally, elicited high-titer anti-OspA-specific antibody that provided protection from live spirochete challenge and was sustained over the lifetime of the animal. Both humoral and cell-mediated Th(1) immunity was induced, as shown by anti-OspA immunoglobulin G2a antibody and preferential gamma interferon production by OspA-specific CD4(+) T cells.

FIG. 1.

(a) Time course of induction of viral NS1 (83 kDa) and NS2 (24 kDa) expression following infection by MVMi of primary C3H/HeNcr splenocytes either unstimulated, or stimulated by solid-phase cross-linking of murine CD3e (T-cell receptor), as described in the text. Proteins were detected by Western blot with an antibody specific for the N-terminal region common between NS1 and NS2. (b) Time course of induction of VP1 (83 kDa) and VP2 (64 kDa) expression in splenocyte cultures treated as in panel a. Proteins were detected by Western blot with an antibody specific for denatured capsid protein.

FIG. 2.

(a) Sequence organization of transducing/vector and helper/packaging plasmids. OspA transgene insertion is shown in green, MVM sequences are in red (NS) or blue (VP), and LuIII is in purple. The positions of the viral early (P4) and late (P38) promoters are indicated. Amb indicates the position of a translation termination codon engineered into the VP1 coding sequence in order to abolish its expression. Simian virus 40 origin of replication and simian virus 40 polyadenylation sites in the packaging construct are shown as gray ovals and arrowheads, respectively. The rabbit β-globin polyadenylation site is shown in yellow. Panels b through f show immunofluorescent staining for NS1 (b and d) and OspA (c and e) in human NB324K cells (b and c) and anti-CD3-stimulated murine splenocytes (d and e) transduced with MVMi-OspA vector, stained at 48 h postinoculation Panel f shows a Western blot for NS1 (83 kDa) and OspA (30 kDa) expressed in vector-transduced murine splenocytes 48 h after stimulation by CD3 cross-linking.

FIG. 3.

ELISAs of anti-OspA serum antibody for individual C3H mice (•, ✶, ♦, and □) following a single intravenous vaccination with 10⁶ transducing units of MVMi-OspA vector per mouse, obtained at the indicated times postinoculation. MVMi-EGFP vector-immunized animals were followed as a control (▪).

FIG. 4.

Photomicrographs of hematoxylin- and eosin-stained tissue sections of tibiotarsus (a and b) and heart (c and d) taken from MVMi-EGFP vector-immunized control mice (a and c) and from MVMi-OspA vector-immunized animals (b and d). The mice were challenged 7 weeks after immunization and tissues were prepared 2 weeks later. Arrows indicate regions of bone erosion. Insets are at 10-fold-higher magnification.

FIG. 5.

Results from ELISAs of serum antibodies obtained at the indicated times from C3H/HeNcr mice following a single vaccination with the MVMi-OspA vector by the intravenous (○), intranasal (▪), and subcutaneous (♦) routes and with MVMi-EGFP vector administered intravenously (✶). Each point represents the geometric mean of titers for five mice assayed individually.

FIG. 6.

(a) Histogram of anti-OspA immunoglobulin isotypes induced by different inoculation routes. Sera were obtained from five individuals at 3 weeks following inoculation. (b) Scattergram of IL-4 and IFN-γ secretion by CD4⁺ cells purified at 9 or 12 months after inoculation from individual spleens of MVMi-OspA vector-immunized or EGFP-vaccinated control mice and stimulated by coculture with naive splenocytes presenting lipidated OspA.