Vaccinia Virus H3L Envelope Protein Is a Major Target of Neutralizing Antibodies in Humans and Elicits Protection against Lethal Challenge in Mice

Abstract

The smallpox vaccine is the prototypic vaccine, yet the viral targets critical for vaccine-mediated protection remain unclear in humans. We have produced protein microarrays of a near-complete vaccinia proteome and used them to determine the major antigen specificities of the human humoral immune response to the smallpox vaccine (Dryvax). H3L, an intracellular mature virion envelope protein, was consistently recognized by hightiter antibodies in the majority of human donors, particularly after secondary immunization. We then focused on examining H3L as a valuable human antibody target. Purified human anti-H3L antibodies exhibited substantial vaccinia virus-neutralizing activity in vitro (50% plaque reduction neutralization test [PRNT 50 ] ‫؍‬ 44 g/ml). Mice also make an immunodominant antibody response to H3L after vaccination with vaccinia virus, as determined by vaccinia virus protein microarray. Mice were immunized with recombinant H3L protein to examine H3L-specific antibody responses in greater detail. H3L-immunized mice developed hightiter vaccinia virus-neutralizing antibodies (mean PRNT 50 ‫؍‬ 1:3,760). Importantly, H3L-immunized mice were subsequently protected against lethal intranasal challenges with 1 or 5 50% lethal doses (LD 50 ) of pathogenic vaccinia virus strain WR, demonstrating the in vivo value of an anti-H3L response. To formally demonstrate that neutralizing anti-H3L antibodies are protective in vivo, we performed anti-H3L serum passive-transfer experiments. Mice receiving H3L-neutralizing antiserum were protected from a lethal challenge with 3 LD 50 of vaccinia virus strain WR (5/10 versus 0/10; P < 0.02). Together, these data show that H3L is a major target of the human anti-poxvirus antibody response and is likely to be a key contributor to protection against poxvirus infection and disease.

Dominance and Diversity in the Primary Human CD4 T Cell Response to Replication-Competent Vaccinia Virus

“…H3L was strongly recognized by subjects 2 and 10. This protein induces neutralizing Abs (49,72). H3L 229 -237 (ILDNAAKYV) accounted for ϳ7% of reactivity to whole virus (Fig.…”

“…Several potent vaccinia neutralizing membrane Ags have been described (38,39,(47)(48)(49). We expressed and purified three proteins (L1R, A33R, B5R) as extracellular domains and one (A27L) as a full-length construct.…”

Transcriptionally active PCR for antigen identification and vaccine development: In vitro genome-wide screening and in vivo immunogenicity

“…This study represents, to the best of our knowledge, the first demonstration of the use of transcriptionally active PCR products or any linear PCR fragments as a useful high throughput tool for screening genomic sequence data to identify potential target antigens for vaccine development. Recently, proteinbased approaches have proved promising in this regard [37,38], We further show that transcriptionally active PCR products or linear PCR fragments can be immunogenic in vivo, and are capable of inducing both antibody and cellular immune responses at levels equivalent to those induced by the corresponding supercoiled plasmid DNA. Thus, a TAP-based approach allows for the relative prioritization amongst target antigens included in a high throughput genomic screening strategy on the basis of their immune reactivity.…”

GRA1 protein vaccine confers better immune response compared to codon-optimized GRA1 DNA vaccine

“…Protein microarray chips were prepared as described [48,51]. Briefly, 15 l purified rGRA1 (2.5 mg/ml) printed onto nitrocellulose-coated FAST TM glass slides (Schleicher & Schuell, Keene, NH) using an OmniGrid 100 microarray printer (Genomics Solutions, Ann Arbor, MI).…”