DNA vaccination in utero: a new approach to induce protective immunity in the newborn

Abstract

Infectious diseases are the primary cause of neonatal morbidity and mortality in people, resulting in millions of deaths every year. Infection of the newborn with some of the pathogens involved, such as herpes simplex virus (HSV), human immunodeficiency virus (HIV), hepatitis B virus (HBV), human cytomegalovirus (HCMV) or group B Streptococcus sp. (GBS), usually occurs at the end of pregnancy, during birth or by breast feeding. Therefore, active immunization of the fetus might represent an effective approach to reduce the high risk of neonatal diseases. We recently showed that DNA immunization in utero within the third trimester of gestation induced strong humoral and cell-mediated immune responses in immunized fetal lambs. Here, we demonstrate that fetal immunization was safe and did not affect fetal gestation, neonatal viability, or significantly alter blood leukocyte populations. In utero immunization resulted in the induction of protective mucosal immunity and immune memory in the newborn lamb. Furthermore, there was no evidence that in utero DNA immunization induced immune tolerance. Our results also indicate that the uptake and expression of the plasmid DNA already occurred within the epithelium of the oral cavity. This correlates with our previous findings that local immune responses were found exclusively in the retropharyngeal lymph nodes draining the oral cavity.

Introduction

Early life infections with pathogens such as herpes simplex virus (HSV), human immunodeficiency virus (HIV), hepatitis B virus (HBV), group B Streptococcus sp. (GBS), Haemophilus sp. or Chlamydia sp. are responsible for significant morbidity and mortality in large numbers of human infants every year (reviewed in [1], [2]). Disease transmission from mother to infant frequently occurs shortly before, during or after birth by early rupture of the amniotic membranes or direct contact with infectious secretions during labor. Infections can also result from non-sterile delivery techniques, by breast feeding or during the first days of life in a perinatal nursery. To reduce the risk of infection, improved neonatal care together with caesarian sections, antibiotic treatments, and maternal antiviral therapy are currently being used to prevent vertical disease transmission. However, there is a significant need for alternative therapeutic approaches to prevent neonatal infection.

Vaccination has provided a very cost-effective approach to prevent infectious diseases. However, the induction of tolerance or a state of non-responsiveness was previously thought to preclude vaccination as an effective therapy in the fetus or newborn [3]. We recently demonstrated that DNA immunization in utero induced strong immunity in fetal lambs when delivered into the oral cavity into the amniotic fluid at days 120–125 of gestation [4], [5] (gestation period of sheep is 145 days). Strong humoral and cell-mediated immune responses were found in all immunized fetuses following a single administration of 500 μg plasmid DNA encoding the truncated version of glycoprotein gD of bovine herpesvirus-1 (BHV-1). Additionally, antigen (Ag)-specific antibody-secreting cells were found in the retropharyngeal lymph node draining the oral cavity. Thus, we showed for the first time that in utero immunization with plasmid DNA induces strong immunity in the fetus, which could have a significant impact on survival and quality of life for the large number of infants infected during or shortly after birth [4], [5]. However, since manipulations in utero could result in abortions or fetal misdevelopment, we addressed the safety of in utero DNA vaccination by analyzing fetal development, neonatal viability and development of the neonatal immune system.

Fetal lambs represent a valuable model for analyzing in utero vaccination because of a number of physiological similarities between the ovine and human fetal development and the ontogeny of their immune systems. Using fetal lambs we confirmed observations in higher primates where fetal immunization with a recombinant protein vaccine induced active immunity in the newborn [6]. Three in utero immunizations with a hepatitis B (HB) vaccine induced detectable immune responses in 75% (5/8) of newborn baboons [6]. Thus, these observations verify that the last term fetus is fully immunocompetent and that in utero immunization may represent a useful approach to prevent vertical disease transmission in diseased pregnant women. Vertical disease transmission occurs primarily through mucosal surfaces and we therefore analyzed the induction of mucosal immunity and immune memory in the newborn by in utero immunization which would significantly enhance disease protection in the neonate. To identify the sites of uptake and expression of the plasmid DNA we used luciferase encoding plasmid DNA for in utero delivery and analyzed local expression within the oral cavity and other mucosal sites. Neonatal immune memory was addressed by analyzing immune responses to either a secondary DNA vaccination or a viral infection within the first 10 days after birth. Moreover, immune protection against a respiratory viral infection was assessed in an experimental challenge with infectious BHV-1. To compare the efficacy of fetal DNA immunization with a licensed protein vaccine, we analyzed the induction of protective immunity against HBV, an important pathogen of newborn infants (reviewed in [7], [8]) following fetal immunization with either a HBV surface antigen (HBsAg) protein or DNA vaccine.