Homologous and Heterologous Immunization with a PIV5-Based Modified OspA Vaccine Confers Equivalent Protection Against Tick-Transmitted <i>Borrelia burgdorferi</i>

Vaccines targeting outer surface protein A (OspA) of <i>Borrelia burgdorferi</i> protect against Lyme disease by inducing antibodies in the host that neutralize spirochetes in the <i>Ixodes scapularis</i> tick midgut during engorgement before transmission occurs. We evaluated whether heterologous vaccination enhances protection compared to homologous delivery of the immunogen. C3H-HeN mice were immunized with a parainfluenza virus 5 vector (PIV5) containing a modified OspA protein (OspA _BPBPk ) using three prime-boost immunization regimens: homologous PIV5 intranasal/intranasal (IN/IN), homologous rOspA _BPBPk (protein) subcutaneous/subcutaneous (SC/SC), or heterologous intranasal PIV5-A _BPBPk /subcutaneous rOspA _BPBPk (IN/SC). Immunized mice were then challenged with nymphal <i>I. scapularis</i> ticks infected with 19 strains of <i>B. burgdorferi</i> three months post-prime vaccination. Three weeks after the last day of tick challenge, blood and tissues were collected from euthanized mice. All OspA-containing regimens elicited strong systemic IgG antibody responses that exceeded established protective thresholds. Vaccination markedly reduced <i>B. burgdorferi</i> loads in engorged nymphal ticks. Homologous IN/IN and SC/SC regimens produced the lowest geometric mean <i>flaB</i> burdens in nymphs (2.6 × 10 ³ and 1.8 × 10 ³ copies, respectively), corresponding to ∼1.8-2.0 log ₁₀ reductions relative to controls; the heterologous IN/SC regimen produced a more modest reduction (∼1.7 log ₁₀ ; p = 0.0071 vs IN/IN, p = 0.0003 vs SC/SC). Across all vaccinated groups, no systemic infection with <i>B. burgdorferi</i> was observed as evidenced by absence of motile spirochetes in cultures from tissues, although one mouse (1/9, 11%) in the heterologous IN/SC regimen, had evidence of increased pepVF seroconversion and low-level <i>flaB</i> DNA in culture. Thus, homologous regimens yielded more consistent protective immunity with absent of signs of <i>B. burgdorferi</i> dissemination, suggesting that high systemic anti-OspA _BPBPk IgG antibody titers, rather than alternate immunization routes, were associated with the most consistent protection outcomes. PIV5-A _BPBPk is a promising vaccine candidate for development of next-generation homologous or heterologous human Lyme disease vaccines.