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. 2005 Sep;79(18):11724-33.
doi: 10.1128/JVI.79.18.11724-11733.2005.

Vaccinia virus H3L envelope protein is a major target of neutralizing antibodies in humans and elicits protection against lethal challenge in mice

D Huw Davies  1 , Megan M McCausland, Conrad Valdez, Devan Huynh, Jenny E Hernandez, Yunxiang Mu, Siddiqua Hirst, Luis Villarreal, Philip L Felgner, Shane Crotty
Affiliations

Vaccinia virus H3L envelope protein is a major target of neutralizing antibodies in humans and elicits protection against lethal challenge in mice

D Huw Davies et al. J Virol. 2005 Sep.

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 high-titer 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 [PRNT50] = 44 microg/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 high-titer vaccinia virus-neutralizing antibodies (mean PRNT50 = 1:3,760). Importantly, H3L-immunized mice were subsequently protected against lethal intranasal challenges with 1 or 5 50% lethal doses (LD50) 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 LD50 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.

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Figures

FIG. 1.
FIG. 1.
Protein microarray analysis of human antibodies generated during vaccinia infection reveals dominance of H3L. (A) VV proteome microarray showing representative antibody profile of a donor undergoing a secondary vaccinia virus infection. This donor was immunized 40 years previously and boosted with Dryvax; serum for probing the array was obtained 30 days after the boost. Annotated spots are vaccinia virus-specific antibody reactivities, whereas nonannotated signals are irrelevant non-VV “background” antibody reactivities that are also seen in Dryvax-naïve human sera (15). (B) Detail of proteome microarrays showing spots for H3L and control protein (nonexpressing plasmid) probed with sera from human donors before (Pre) and after (Post) Dryvax immunization. The data shown are representative of primary (1°) and secondary (2°) infections by a vaccinia virus-naive individual and a different previously vaccinated donor, respectively. In both cases, sera were taken 30 days after a Dryvax immunization. The previously vaccinated donor shows a typical anamnestic response to H3L. VIG is shown for comparison. Neutralization titers (PRNT50) were as follows: naive donor, pre = <1/10, post = 1/2,000; previously vaccinated, pre = 1/40, post = 1/3,000; VIG > 1/15,000. (C) Quantification of anti-H3L and control signal intensities from the arrays shown in panel B. (D) Immunoblot of H3L expressed in vitro, probed with the same sera used in panel B. The location of vaccinia virus H3L among the E. coli bands was determined by anti-histidine tag antibody (His). In arrays and immunoblots, sera were used at 1/50 dilution, whereas VIG was used at 1/500.
FIG. 2.
FIG. 2.
Human anti-H3L antibodies neutralize vaccinia virus in vitro. (A) Representative immunoblot of whole vaccinia virus particles used for screening affinity-purified anti-H3L antibodies. VIG control depl., VIG control depleted against E. coli proteins; VIG H3L depl., VIG depleted against H3L protein; VIG anti H3L, human anti-H3L antibodies affinity purified from VIG. The right middle lane unequivocally determines the location of the H3L band. (B) Plaque neutralization assays of monospecific human anti-H3L antibodies affinity purified from VIG. The PRNT50 of purified human anti-H3L was 44 μg/ml. One of three independent experiments is shown. Neutralization of VV by anti-H3L was statistically significant (P < 0.0001).
FIG. 3.
FIG. 3.
Mice immunized with vaccinia virus develop an immunodominant anti-H3L response. (A) Representative scan of a vaccinia virus proteome microarray probed with sera from an individual BALB/c mouse (no. 80) immunized i.p. 21 days previously with VVWR. The identities of these antigens have been described previously (15). Arrays probed with preimmune sera showed no reactivity in this array (15). (B) Quantification of arrays stained with sera from six individual mice immunized 21 days previously with VVWR illustrating the consistency of the responses. Underlined proteins are known envelope proteins. The horizontal line is a stringent cutoff, defined as the mean control signals plus three times the standard deviation. H3L is the strongest VV envelope protein recognized.
FIG. 4.
FIG. 4.
Mice immunized with H3L protein develop potent anti-VV neutralizing antibodies. (A) Immunization schematic. Mice were immunized twice with H3L protein in adjuvant, with the immunizations spaced 3 weeks apart. Control groups of mice received injections of adjuvant alone, or no injections, or a single i.p. immunization with 105 PFU VVNYBOH. The mice were monitored for anti-H3L responses at multiple time points postimmunization. 1°, primary; 2°, secondary. (B) Representative array scan of serum from a single mouse immunized with recombinant H3L adjuvanted in Ribi. (C) Representative immunoblot of serum from a single mouse immunized against recombinant H3L protein (left lane) probed against whole vaccinia virus particles (Fig. 2A). The right lane shows affinity-purified human anti-H3L to localize the H3L band. (D) Anti-VV IgG in H3L-immunized mice was quantified by ELISA. Sera from H3L-immunized mice were tested against VV-infected cell lysate. Anti-VV IgG levels were significantly above baseline after primary (P < 0.01; endpoint titer = 1,366) and secondary (P < 0.001; endpoint titer = 1.27 × 106) immunizations with H3L. (E) Anti-VV neutralizing-antibody titers (PRNT50) were measured in H3L-immunized mice and mice immunized with VVNYBOH. Mean neutralizing antibody titers in H3L-immunized mice were 3,760. Mean neutralizing antibody titers in 105 PFU VVNYBOH-immunized mice were 172.
FIG. 5.
FIG. 5.
Protection of mice immunized with recombinant H3L protein against an intranasal challenge with VVWR. (A) Immunization and challenge protocol schematic. Mice were immunized twice with H3L protein in adjuvant, with the immunizations spaced 3 weeks apart. Control groups of mice received injections of adjuvant alone (first experiment only), or no injections, or a single immunization with 105 PFU VVNYBOH. 1°, primary; 2°, secondary. (B) Body weight was tracked after intranasal challenge with 1 LD50 of VVWR. H3L-immunized mice exhibited significantly less weight loss (P < 0.01) than group A and D control mice (unimmunized). No difference was observed between groups A and D (not shown). Groups A and D combined as “unimmunized,” n = 12; VVNYBOH, n = 4; H3L, n = 4. (C) Survival curve after intranasal challenge with 1 LD50 of VVWR. Approximately 50% of the unimmunized mice died from the intranasal VVWR infection, while all H3L-immunized mice survived (P < 0.02). (D) Body weight was tracked after intranasal challenge with 5 LD50 of VVWR. Weight loss was less severe in the H3L-immunized cohort (P < 0.01). (E) Survival curve after intranasal challenge with 5 LD50 of VVWR. H3L-immunized mice exhibited substantial protection from death compared to unimmunized mice (P < 0.02; 4/5 H3L, 1/7 unimmunized).
FIG. 6.
FIG. 6.
Passive immunization with anti-H3L neutralizing antibodies provides partial protection from a lethal VVWR challenge. (A) VV neutralization activity of rabbit anti-H3L serum. (B) Survival curve after 3-LD50 VVWR intranasal challenge. Ten mice per group. H3L-immunized mice were protected (P < 0.02).
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