Development of a highly efficacious vaccinia-based dual vaccine against smallpox and anthrax, two important bioterror entities

Abstract

Bioterrorism poses a daunting challenge to global security and public health in the 21st century. Variola major virus, the etiological agent of smallpox, and Bacillus anthracis, the bacterial pathogen responsible for anthrax, remain at the apex of potential pathogens that could be used in a bioterror attack to inflict mass casualties. Although licensed vaccines are available for both smallpox and anthrax, because of inadequacies associated with each of these vaccines, serious concerns remain as to the deployability of these vaccines, especially in the aftermath of a bioterror attack involving these pathogens. We have developed a single vaccine (Wyeth/IL-15/PA) using the licensed Wyeth smallpox vaccine strain that is efficacious against both smallpox and anthrax due to the integration of immune-enhancing cytokine IL-15 and the protective antigen (PA) of B. anthracis into the Wyeth vaccinia virus. Integration of IL-15 renders Wyeth vaccinia avirulent in immunodeficient mice and enhances anti-vaccinia immune responses. Wyeth/IL-15/PA conferred sterile protection against a lethal challenge of B. anthracis Ames strain spores in rabbits. A single dose of Wyeth/IL-15/PA protected 33% of the vaccinated A/J mice against a lethal spore challenge 72 h later whereas a single dose of licensed anthrax vaccine protected only 10%. Our dual vaccine Wyeth/IL-15/PA remedies the inadequacies associated with the licensed vaccines, and the inherent ability of Wyeth vaccinia virus to be lyophilized without loss of potency makes it cold-chain independent, thus simplifying the logistics of storage, stockpiling, and field delivery in the event of a bioterror attack involving smallpox or anthrax.

The 2001 terror attacks in the United States have heightened the potential threat of bioterrorism in the United States and around the globe. Because of the purported past use of Variola major and Bacillus anthracis in biowarfare and the reported possible existence of military programs for weaponization of these agents by some nations and rogue regimes, smallpox and anthrax represent the most deadly bioterror entities (1–3) that are a threat to our national security and public health. Therefore, the development of medical countermeasures against their potential use remains a national priority (4).

Smallpox is a highly communicable, often fatal disease with mortality rates approaching 30% in susceptible individuals (5). Currently there is no approved treatment for smallpox although vaccination within the first few days of exposure may provide some level of protection against the disease (6) (http://www.bt.cdc.gov/agent/smallpox/vaccination/facts.asp). Variola virus, the etiological agent of smallpox, is a member of the Orthopoxvirus subfamily of viruses, which also includes the vaccinia virus that induces potent cross-protective immunity against Variola virus. The medical triumph of eradicating smallpox as a natural disease in 1977 was achieved with the use of a calf-lymph–derived live vaccinia virus vaccine (Dryvax containing the Wyeth strain in the United States), thus attesting to the efficacy and immunogenicity of vaccinia viruses against Variola virus. In the United States, smallpox vaccinations in the civilian population ceased over 30 y ago, and we are now faced with an ever-increasing naive population, along with declining immunity in the older generation who were vaccinated in their childhood. Although the currently available licensed vaccine [ACAM 2000 (Acambis, Inc.), a cell-culture-grown derivative (7) of the earlier Dryvax vaccine used in the smallpox eradication campaigns] is highly effective, it can cause serious complications in some vaccinated individuals, including encephalitis, progressive vaccinia, myocarditis, or myopericarditis. Additionally, its use is contraindicated in patients with atopic skin diseases and immune deficiencies and during pregnancy. Unlike 3–4 decades ago, ≈25% of the individuals in contemporary populations are estimated to represent “at-risk groups” for whom live ACAM 2000 vaccine is contraindicated (7). Thus, a safer vaccine that can match the efficacy of Dryvax/ACAM 2000 vaccine but that is devoid of its residual virulence is urgently needed for contemporary populations.

Anthrax caused by the bacterial pathogen B. anthracis clinically manifests itself in cutaneous, gastrointestinal, and inhalational forms, all of which can be fatal, although the inhalational form causes the highest mortality (8). A number of antibiotics such as ciprofloxacin and doxycycline are very effective in eliminating vegetative bacilli, but the ability of the antibiotic-resistant spores to remain dormant in the tissues (9) and subsequently reactivate to cause fatal disease increases the difficulty of treating this disease with antibiotics alone. In fact, even with antibiotic coverage, mortality could be as high as 60–70% especially with the inhalation form. The pathogenesis of anthrax is driven by plasmid-borne virulence factors that include the poly-γ-d-glutamic acid capsule and the tripartite exotoxin that consists of protective antigen (PA), lethal factor (LF), and edema factor (EF). PA plays a central role in virulence by mediating the entry of LF and EF into cells. Collectively, these virulence factors promote the overwhelming systemic pathophysiology associated with B. anthracis infection, often leading to a fatal outcome. The available evidence supports the notion that induction of antibodies solely against PA can be protective against anthrax (10). PA is also the major component of the anthrax vaccine licensed for human use in the United States as an aluminum hydroxide-adsorbed cell-free filtrate of an acapsular strain (V770-NP1-R) of B. anthracis [Biothrax or anthrax vaccine adsorbed (AVA)]. In addition to concerns regarding adverse effects associated with this vaccine, its undefined composition, lot-to-lot variation, and the requirement for multiple doses over a protracted period to achieve adequate levels of protective immunity make this vaccine less than optimal for use in response to a bioterrorism incident. Furthermore, the limited shelf life of ≈4 y of Biothrax/AVA results in the need for periodic replenishment of vaccine in the strategic national stockpile. Therefore, a compelling need exists for a better vaccine against B. anthracis that can confer rapid immunity with an abbreviated immunization schedule that can be stored long term and deployed quickly in the event of a bioterror event.

We recently reported that the integration of IL-15, a cytokine with pleiotropic immune modulatory activities, into the Wyeth strain of vaccina virus derived from the licensed smallpox vaccine resulted in the development of a smallpox vaccine candidate (Wyeth/IL-15) with superior efficacy and immunogenicity. This smallpox vaccine candidate out-performed the parental vaccine in two animal models that sufficiently recapitulate the pathophysiology of smallpox in humans; mice were protected when lethally challenged with a neurotropic strain of vaccinia virus intranasally and cynomolgus monkeys were protected from a lethal i.v. challenge of monkeypox virus (11, 12). Moreover, unlike the parental virus, our Wyeth/IL-15 is nonlethal in T-cell–deficient athymic nude mice at a dose of 10⁷ plaque-forming units (pfu) and undergoes enhanced clearance in these mice, attesting to its safety even in immunocompromised hosts (12). Having demonstrated the superiority in immunogenicity and attenuation of virulence of IL-15–integrated Wyeth vaccinia, we exploited our Wyeth/IL-15 platform to generate a dual vaccine effective against both smallpox and anthrax by integrating the PA gene of B. anthracis (Wyeth/IL-15/PA) to overcome the problems of poor immunogenicity and apparent lack of immunological memory associated with the licensed Biothrax/AVA vaccine. We demonstrate that Wyeth/IL-15/PA confers superior protection against inhalation anthrax in both mice and rabbits and make a direct comparison with the licensed Biothrax/AVA vaccine. We believe our dual vaccine, Wyeth/IL-15/PA, which is effective against two of the most deadly pathogens, will help consolidate and simplify our national bioterror counterefforts by streamlining the manufacture, stockpiling, and swift deployment of such vaccines should the need arise.

Results

Validation of Dual Vaccine Agent for the Expression of Molecular Adjuvant IL-15 and PA of B. anthracis.

The integration of human IL-15 and B. anthracis PA genes in a head-to-tail configuration into the hemagglutinin locus of the licensed smallpox vaccine strain (Wyeth) of vaccinia resulted in a stable recombinant virus (Wyeth/IL-15/PA) that displayed no phenotypic or growth characteristic differences in comparison with the parental virus. The presence of human IL-15 in the culture supernatants of Wyeth/IL-15/PA–infected cells was confirmed by a human IL-15–specific ELISA, and a CV-1 cell monolayer infected at a multiplicity of infection of 10 yielded 2–4 ng/mL of IL-15 in the culture supernatant 3 d postinfection, which is consistent with the levels that we previously reported for Wyeth/IL-15 (11). Because there is considerable interest in nonreplicative vaccinia viruses such as the modified vaccinia virus Ankara (MVA) as an alternate smallpox vaccine that can be safely administered to individuals for whom the use of the licensed Wyeth strain vaccinia is contraindicated, and because we have shown earlier that IL-15–integrated MVA (MVA/IL-15) is an effective vaccine in two animal models that recapitulate smallpox in humans (11, 12), we generated a PA-expressing recombinant virus in the MVA/IL-15 backbone as well (MVA/IL-15/PA). As shown in Fig. 1, an abundant expression of PA was confirmed by immunoblot analyses of Wyeth/IL-15/PA or MVA/IL-15/PA–infected BHK-21 cell lysates. It should be noted that the apparent molecular weight of the vaccinia-expressed PA in BHK-21 cells is higher than the predicted 83 kDa. The primary amino acid sequence of the PA polypeptide contains multiple potential N-linked and O-linked glycosylation sites that could add carbohydrate moieties as has been reported previously (13, 14). Thus we were able to reduce the molecular weight of vaccinia-expressed PA to be closer to the predicted size of 83 kDa by treating the PA with PNGase F, which cleaves sugars from asparagine residues (Fig. 1).

Fig. 1.

The expression of glycosylated B. anthracis PA by recombinant vaccinia viruses with an integrated IL-15 gene. BHK-21 cells were infected with Wyeth/IL-15/PA and MVA/IL-15/PA for 48 h, and the infected BHK-21 cell lysates were subjected to SDS/PAGE on a 10% acrylamide gel. Western blot analyses were performed using a polyclonal goat PA-specific antibody (List Biological Laboratories). The glycosylation of vaccinia virus-expressed PA polypeptide in BHK21 cells was assessed by the treatment of vaccinia-infected cell lysates with PNGase F enzyme (New England Biolabs) before SDS/PAGE. The positions of molecular weight markers are on shown on left.

Immunogenicity and Efficacy of Wyeth/IL-15/PA in Rabbits.

To assess the immunogenicity and efficacy of Wyeth/IL-15/PA in comparison with the licensed Biothrax/AVA, groups of female New Zealand White rabbits (each group consisting of nine animals) were vaccinated with the respective vaccines. As a negative control, a group of six animals were vaccinated with Wyeth/IL-15 (see Table 1). When we measured the anti-PA ELISA antibody titers in the serum samples from blood collected 28 d post primary vaccination, immediately before administering the booster dose of the vaccine the mean PA-binding antibody titer for the Wyeth/IL-15/PA group was 46,220 whereas for the group of rabbits that received the Biothrax/AVA the mean titer was 16,220 and the difference was statistically significant (P = 0.038). The same trend was noted when we examined the serum samples collected 21 d after the booster vaccination; immediately before animals were subjected to an inhalation challenge with a mean dose of 262 LD₅₀ of B. anthracis Ames strain spores, we noted a mean anti-PA ELISA titer of 515,600 for the group vaccinated with Wyeth/IL-15/PA in comparison with a mean titer of 275,600 for the Biothrax/AVA group (P = 0.012). These results indicate that Wyeth/IL-15/PA displays superior immunogenicity in rabbits in a direct comparison with the licensed Biothrax/AVA when administered in a single-dose vaccination regimen or a two-dose vaccination regimen. However, surprisingly, when we determined the toxin-neutralizing antibody titer (TNA) in the serum samples collected 21 d after the booster vaccination for the two groups (Table 1), we noted a discordance in which the mean TNA titer for the Wyeth/IL-15/PA group was 4.5-fold lower than that of the Biothrax/AVA group (478 versus 2153), which was statistically significant (P = 0.009). This observation is important because, for almost all reported PA-based vaccine candidates, the vaccine-induced TNA titers paralleled those of PA-binding ELISA titers quantitatively (16–18). Our results may reflect the possibility that vaccinia-expressed, glycosylated PA induces a qualitatively different humoral immune response in vaccinated rabbits. Mirroring our observation, in a recent study Livingston et al. (19) reported that, whereas electroporation-mediated delivery of a DNA-based PA vaccine in rhesus monkeys elicited a higher PA-binding ELISA titer than Biothrax/AVA, the TNA titers induced by the DNA vaccine were fourfold less than the titers induced by Biothrax/AVA vaccine. It is important to note that, in the present study, the protection of vaccinated rabbits from a lethal intranasal Ames spore challenge was achieved without any demonstrable bacterimia. Thus, it is likely that protection occurs at a stage before vegetative bacterial multiplication and copious toxin production with the implication that the anti-spore activity of PA-specific antibodies, and not necessarily the toxin-neutralizing activity of anti-PA antibodies, is the dominant component in mediating the sterile protection seen in our study, which is similar to the observations of Welkos et al. (20) and Stepanov et al. (21).

Table 1.

Anthrax vaccine study in rabbits with two doses of vaccine

		Anti-PA ELISA titer			Anti-vaccinia titer†
Experimental group	Identification tag no.	Prime	Boost	TNA titer*	Prime	Boost	Postchallenge survival	Postchallenge anti-PA ELISA titer	Postchallenge bacteremia
Wyeth/IL-15/PA	38	32,000	640,000	350.8	100	800	Yes	2560,000	Negative
	34	128,000	320,000	397.7	400	800	Yes	640,000	Negative
	54	64,000	640,000	840.6	100	1,600	Yes	1,280,000	Negative
	48	64,000	640.000	448.4	400	1,600	Yes	640,000	Negative
	55	16,000	640,000	106.1	<100	800	Yes	2,560,000	Negative
	27	16,000	320,000	291.8	<100	400	Yes	1,280,000	Negative
	51	32,000	640,000	1,326.5	<100	800	Yes	640,000	Negative
	40	32,000	160,000	128.7	100	800	Yes	640,000	Negative
	47	32,000	640,000	1,628.8	100	800	Yes	2,560,000	Negative
Biothrax/AVA	41	8,000	160,000	1,186.8	ND	ND	Yes	640,000	Negative
	44	<2,000	80,000	593.1	ND	ND	Yes	1,280,000	Negative
	42	8,000	320,000	4,519.0	ND	ND	Yes	1,280,000	Negative
	25	64,000	320,000	2,103.0	ND	ND	Yes	640,000	Negative
	37	16,000	160,000	1,405.8	ND	ND	Yes	320,000	Negative
	33	16,000	160,000	2,360.0	ND	ND	Yes	640,000	Negative
	56	8,000	320,000	1,074.5	ND	ND	Yes	1,280,000	Negative
	39	16,000	320,000	1,985.8	ND	ND	Yes	1,280,000	Negative
	52	8,000	640,000	2,306.3	ND	ND	Yes	2,560,000	Positive
Control vaccinia	31	ND	ND		<100	800	No	ND	Positive
	24	ND	ND		100	800	No	ND	Positive
	49	ND	ND		<100	1,600	No	ND	Positive
	30	ND	ND		100	800	No	ND	Positive
	36	ND	ND		100	800	No	ND	Positive
	29	ND	ND		<100	800	No	ND	Positive

Animals were vaccinated twice 4 wk apart. Recombinant vaccinia vaccines were given s.c. at a dose of 1 x 10⁷ pfu. Biothrax/AVA vaccine was given at a dose of 50 μL/animal intramuscularly. For serological assays, sera were collected 4 wk after primary vaccination (“Prime”), 3 wk after booster vaccination (“Boost”), or 14 d after spore challenge. Inhalation challenge of B. anthracis (Ames) spores (∼200 LD₅₀) was done 3 wk after booster vaccination. Postchallenge survival was monitored for 14 d. Postchallenge bacteremia was assessed 6 d after spore challenge. ND. not detected.

*Serum samples were collected 3 wk after booster vaccination, and LT toxin-neutralizing antibody titers were determined using J774.1 as described previously (15).

^†Vaccinia virus-neutralizing antibody titers were determined by a plaque reduction assay where reduction of 80% of the in-put virus was taken as the anti-vaccinia titer.

As a prerequisite for being an effective dual vaccine against both anthrax and smallpox, we confirmed the ability of Wyeth/IL-15/PA to elicit robust vaccinia virus-neutralizing antibodies that were comparable to the levels generated by Wyeth/IL-15, a superior smallpox vaccine that we reported previously (11). Notably, the induction of vaccinia-neutralizing antibodies with the first dose of Wyeth/IL-15/PA did not have a negative impact on the booster vaccination given 4 wk later in enhancing vaccinia-specific neutralizing antibodies and the heterologous PA antigen-directed antibodies, contrary to the commonly held view that circulating anti-vaccinia–neutralizing antibodies could render subsequent revaccinations ineffective.

To ascertain the efficacy of our Wyeth/IL-15/PA vaccine, vaccinated rabbits were challenged 21 d after the second dose of vaccine. Although our intended aim was to achieve a dose of 200 LD₅₀ [1 LD₅₀ = 105,000 colony-forming units (cfu)] of spores from the Ames strain of B. anthracis, the inhalation dosimetry revealed that the group vaccinated with Wyeth/IL-15/PA received a dose of 240 LD₅₀ (2.52 × 10⁷ cfu) whereas the group vaccinated with Biothrax/AVA received a dose of 255 LD₅₀ (2.68 × 10⁷ cfu). The control group vaccinated with Wyeth/IL-15 received a dose of 290 LD₅₀ (3.05 × 10⁷ cfu). Throughout a 14-d observation period after the spore challenge, with daily measurements of body weights and three-times-a-day measurement of body temperature, none of the animals vaccinated with Wyeth/IL-15/PA or Biothrax/AVA showed any appreciable changes in these parameters. Moreover, blood samples taken 6 d after challenge were culture negative for B. anthracis in all nine rabbits vaccinated with Wyeth/IL-15/PA, whereas one rabbit in the Biothrax/AVA group was bacteremic with B. anthracis. However, despite the lack of culture evidence for anthrax infection, when we assessed the serum samples collected 14 d after the spore challenge from the rabbits vaccinated with Wyeth/IL-15/PA and Biothrax/AVA (Table 1), we noted a two- to fourfold rise in PA-specific ELISA antibody titers for most animals, although one cannot interpret this to indicate an active infection. Perhaps, as has been purported, PA polypeptide is present in the spore coat or exosporium (22, 23) and could have triggered an anamnestic response in these animals, leading to a rise in anti-PA titer. In contrast, all six animals vaccinated with Wyeth/IL-15 died of anthrax disease with a mean time to death of 3.5 d. The blood collected at the moribund stage revealed the presence of B. anthracis in all six animals.

Further Characterization of Vaccinia-Based PA Vaccines in A/J Mice.

Having confirmed the potent efficacy of our Wyeth/IL-15/PA vaccine against a lethal inhalation spore challenge with the highly virulent Ames strain of B. anthracis in rabbits, a recognized animal model of human disease, we set out to further characterize the immunogenicity of our vaccines including the MVA derivative, MVA/IL-15/PA, in a more economical model using A/J mice (24). As shown in Fig. 2, when we tested the sera of vaccinated mice 3 wk after vaccination, as we demonstrated earlier in rabbits, both Wyeth/IL-15/PA and MVA/IL-15/PA induced robust anti-PA antibodies in our PA-specific ELISA. In fact, MVA/IL-15/PA appeared consistently slightly more immunogenic than the replicative Wyeth/IL-15/PA. Concordant with the ability to induce a robust immune response, when we subjected the vaccinated mice to an inhalation spore challenge 21 d after the booster vaccination (20 LD₅₀ dose of spores from an acapsular, toxigenic strain of B. anthracis strain 7702), both Wyeth/IL-15/PA and MVA/IL-15/PA conferred efficient protection that was comparable to the licensed vaccine Biothrax/AVA (Table 2). An important attribute of a vaccine intended for individuals with a potential risk of exposure to anthrax is the durability and the persistence of the vaccine-induced immune response. Therefore, we evaluated the levels of PA antibodies in A/J mice 6 mo after vaccination. As shown in Fig. 3, in an ELISA that captures PA-specfic total IgG levels, the MVA/IL-15/PA–induced antibody levels were comparable to those induced by Biothrax/AVA in both duration and magnitude. However, when we examined the subclass specificity of this PA-specfic IgG response, the immune response induced by MVA/IL-15/PA was predominantly of the Th-1 type with IgG2a and IgG2b levels that exceeded the levels of these IgG subclasses induced by Biothrax/AVA, which predominantly induced a Th-2 type immune response. It is noteworthy that anti-PA antibodies can confer protection against a lethal spore challenge without the appearance of vegetative bacteria in the blood stream, for example, as we have shown above in our rabbits vaccinated with Wyeth/IL-15/PA or Biothrax/AVA (Table 1). Because of the purported presence of PA on the spore coat surface, it is possible that PA-bound antibodies, especially of the Th1 type, are able to initiate powerful effector activities such as complement fixation and antibody-dependent cellular cytotoxicity to prevent germination of spores and lead to their destruction through sporucidal or germination inhibitory mechanisms as has been suggested previously (22).

Fig. 2.

Assessment of PA-specific antibodies in A/J mice vaccinated with vaccinia-based dual vaccines. Mice were vaccinated twice with Wyeth/IL-15/PA, MVA/IL-15/PA (1 × 10⁷ pfu/mouse), or Biothrax/AVA (25 μL/mouse) s.c. 4 wk apart, and 3 wk after the second dose of vaccine, animals were bled. Serial dilutions of pooled sera from each group (n = 6) vaccinated with respective vaccinia recombinant vaccines or Biothrax/AVA were then analyzed by a PA-specific ELISA to determine the levels of PA-specific antibody induced by vaccination.

Table 2.

Anthrax vaccine study in A/J mice with two doses of vaccine

Vaccine	No. of survivors/total (%)
MVA/IL-15/PA	10/10 (100)
Wyeth/IL-15/PA	9/10 (90)
Biothrax/AVA	10/10 (100)
Unvaccinated mice	0/10 (0.0)

Mice were vaccinated twice 4 wk apart s.c.. The dose of recombinant vaccinia vaccines was 1 x 10⁷ pfu/mouse and Biothrax/AVA was 25 μL/mouse. Vaccinated mice were challenged via inhalation route with a dose of 20 LD₅₀ using spores of toxigenic acapsular B. anthracis strain 7702 (pXO1+; pXO2−) and monitored for 14 d.

Fig. 3.

Long-term persistence and Ig subclass specificities of PA-specific antibodies induced by vaccinia-based dual vaccines. Groups of A/J mice (n = 6) were vaccinated with two doses of either MVA/IL-15/PA (1 × 10⁷ pfu/mouse) or Biothrax/AVA (25 μL/mouse) s.c. 4 wk apart, and their serum samples were collected 6 mo after the second vaccination. Pooled serum samples from each group were then tested in a PA-specific ELISA to determine the total anti-PA IgG levels as well as anti-PA IgG_2a and anti-PA IgG_2b levels using either total or subclass-specific anti-mouse IgG secondary antibodies in the ELISA.

From a biodefense perspective, it is most critical to generate a vaccine that can be used in a postevent situation and that is capable of inducing a protective immune response rapidly. When we assessed the kinetics of anti-PA antibody induction after a single dose of our vaccinia-based dual vaccines in A/J mice (Fig. 4), a demonstrable antibody response was evident by the third day post vaccination, whereas in mice vaccinated with Biothrax/AVA, the appearance of demonstrable anti-PA antibodies required more than a week. As shown in Fig. 5, the induction kinetics of vaccinia-neutralizing antibodies were equally rapid with our dual vaccine with measurable neutralizing antibodies appearing within 6 days of vaccination. More importantly, and consistent with the superior induction kinetics of PA-specific antibodies with our vaccinia-based dual vaccines, when we lethally challenged mice (20 LD₅₀ dose of spores) 3 d after a single dose of vaccine, ≈33% of the mice that were vaccinated with either MVA/IL-15/PA or Wyeth/IL-15/PA survived whereas only 10% (2 of 20) of the mice vaccinated with Biothrax/AVA withstood the lethal spore challenge (Table 3). This difference is statistically significant (P = 0.0251). Similarly, in a 6-d postvaccination challenge, Wyeth/IL-15/PA again protected 65% of the mice whereas Biothrax/AVA lagged behind with a 35% survival of vaccinated mice. This reflects the superior efficacy and rapidity of immune response of our vaccinia-based dual vaccines in protecting against inhalation anthrax.

Fig. 4.

Superior induction kinetics of PA-specific antibodies after vaccination with vaccinia-based dual vaccines. A group of A/J mice (n = 6) were given a single dose of MVA/IL-15/PA vaccine (1 × 10⁷ pfu/mouse) or Biothrax/AVA (25 μL/mouse). Animals were bled longitudinally at indicated time points following vaccination, and serial dilutions of pooled sera from each group were tested in a PA-specific ELISA to determine the kinetics of PA-specific antibody induction and to quantitate the levels of such PA-specfic antibodies in their sera. Prebleeds taken from each group before vaccination served as a baseline control.

Fig. 5.

Induction kinetics of vaccinia-neutralizing antibodies following vaccination with vaccinia-based dual vaccines. Mice were vaccinated with a single dose of MVA/IL-15/PA vaccine (1 × 10⁷ pfu/mouse), and longitudinal serum samples were collected as described in the legend to Fig. 4. The induction kinetics of vaccinia-neutralizing antibodies and the quantitation of such antibodies were determined using a plaque reduction assay as described in Materials and Methods. Prebleeds taken from each group before vaccination served as a baseline control.

Table 3.

Short-term anthrax vaccine study in A/J mice with one dose of vaccine

	No. of survivors/total (%)
Vaccine	3-d post vaccination challenge	6-d post vaccination challenge
MVA/IL-15/PA	6/19 (31.6)	8/17 (47.1)
Wyeth/IL-15/PA	6//18 (33.3)*	13/20 (65.0)
Biothrax/AVA	2/20 (10.0)	7/20 (35.0)
Unvaccinated mice	0/17 (0.0)	0/18 (0.0)

Mice were vaccinated once s.c.. The dose of recombinant vaccinia vaccines was 1 x 10⁷ pfu/mouse and Biothrax/AVA was 25 μL/mouse. Vaccinated mice were challenged via inhalation route with a dose of 20 LD₅₀ using spores of toxigenic acapsular B. anthracis strain 7702 (pXO1+; pXO2−) and monitored for 14 d.

*Statistically significant, P = 0.0251, compared with Biothrax/AVA.

Discussion

A US congressional commission on the prevention of weapons of mass destruction, proliferation, and terrorism unanimously concluded that, of all means of mass destruction, bioterrorism poses the greatest threat to the global community in the coming years (25). In a sobering assessment early this year, the same commission assigned a failing grade for our nation's efforts to enhance the medical countermeasures to rapidly mitigate or prevent mass casualties arising from a biological attack (http://www.preventwmd.gov/static/docs/report-card.pdf).

Because of their prior use in biowarfare/bioterrorism or the purported past/present existence of military programs for weaponization, V. major virus and B. anthracis remain at the apex of potential pathogens that could be used in a bioterror attack to inflict mass casualties. Although licensed vaccines are available for both smallpox caused by V. major (ACAM 2000) and anthrax caused by B. anthracis (Biothrax/AVA), serious concerns remain as to the deployability of these vaccines in large-scale vaccination efforts, especially in the aftermath of a bioterror attack involving these pathogens. For example, although the smallpox vaccine has been administered to over one billion people in the past and its stellar efficacy against smallpox is considered a gold standard for infectious disease vaccines, the residual virulence of this vaccine can cause devastating complications, including death in immunodeficient individuals or people with atopic skin disease who account for a sizable swath of the contemporary population (7). On the other hand, although a committee convened by the Institute of Medicine of the National Academies concluded in October 2000 that Biothrax/AVA vaccine is effective against all forms of anthrax, the need for a better vaccine was strongly advocated (26).

In developing Wyeth/IL-15/PA, a dual vaccine that is efficacious against two leading deadly pathogens with high bioterror potential, we integrated cytokine IL-15 into a licensed smallpox vaccine with the disruption of the single vaccinia gene hemagglutinin—which does not play an appreciable role in viral pathogenesis or replication (27)—primarily to attenuate the residual virulence of the Wyeth strain of vaccinia. IL-15 is a powerful immunostimulatory cytokine with a wide range of biological activities (28, 29). It is involved in the activation, proliferation, and differentiation of CD8⁺ T cells and NK cells and in the maintenance of CD8⁺ memory T cells in addition to supporting the survival of mature dendritic cells. IL-15 has also been implicated in B-cell proliferation and differentiation as well as in antibody synthesis and secretion along with a role in maintaining serological memory (30). It is likely that the effects of IL-15 on NK cells and innate immune cells facilitate the rapid clearance of vaccinia virus even in immune-deficient hosts, thereby markedly reducing the residual virulence associated with the Wyeth strain. Our previous work has shown that the incorporation of IL-15 into vaccinia-derived vaccines induces high avidity, long-lived antigen-specific memory cytotoxic T lymphocytes and persistent antigen-specific antibody responses, thereby conferring durable effective immunity against vaccine antigens (31, 32). It is indeed remarkable that Wyeth/IL-15/PA induces a PA-specific protective antibody response within 72 h post vaccination as we have shown in this study, highlighting the potential utility of this vaccine in a postevent scenario. The superiority of our vaccinia-based dual vaccine against anthrax as opposed to the poorly protective WR recombinant described by Iacono-Connors (33) is reflective of the use of a codon-optimized synthetic PA gene along with the incorporation of IL-15 as a molecular adjuvant in our vaccine that enhances the kinetics, magnitude, and longevity of vaccine-induced antibody responses.

It is important to emphasize that the vaccinia-based dual vaccine with integrated IL-15 not only is superior in immunogenicity and efficacy in comparison with the currently licensed vaccines against smallpox and anthrax, but also remedies the inadequacies associated with such licensed vaccines. These inadequacies include the residual virulence seen with smallpox vaccines in immune-deficient hosts and the undefined composition and lot-to-lot variation associated with Biothrax/AVA. Wyeth/IL-15/PA, however, is genetically stable and amenable to defined cell culture-based production within the existing vaccine-manufacturing capabilities. Moreover, the inherent ability of Wyeth vaccinia virus to be lyophilized without loss of potency makes our Wyeth/IL-15/PA vaccine cold-chain independent, thus simplifying the logistics of storage, stockpiling, and field delivery in the event of a bioterror attack involving smallpox or anthrax. We believe that these features make Wyeth/IL-15/PA a preferred choice for integrating into our national biodefense preparedness agenda to protect the nation by enhancing the capabilities of rapidly responding to and recovering from a devastating attack involving these bioweapons of mass destruction.

Materials and Methods

Construction of Vaccinia-Based Vaccine Candidates That Express PA Gene of B. anthracis.

The construction of smallpox vaccine candidates Wyeth/IL-15 and MVA/IL-15 has been described earlier (11, 34). A synthetic PA gene was manufactured de novo that encodes a polypeptide identical to the deduced amino acid sequence of the mature PA polypeptide described by Welkos et al. (35) (GenBank accession no. AAA22637.1) with preferred human codons with a signal peptide derived from the tissue plasminogen activator polypeptide along with vaccinia expression sequence elements embedded 5′ for proper expression. In constructing Wyeth/IL-15/PA and MVA/IL-15/PA, the synthetic PA gene was integrated into the hemagglutinin locus such that IL-15 and PA genes were in a head-to-tail configuration at the hemagglutinin locus of the respective recombinant viral genomes. Wyeth/IL-15 and Wyeth/IL-15/PA viral stocks were made and titrated in CV-1 monkey kidney cells, whereas MVA/IL-15/PA stocks were made and titrated in BHK-21 cells.

Measurement of Antibody Titers in Serum Samples from Vaccinated Animals.

An ELISA was used to determine the titer of anti-PA–binding antibodies. Briefly, plates were coated with recombinant PA (List Biological Laboratories) at a concentration of 1 mcg/mL in carbonate coating buffer (pH 9.0) overnight at 4 °C. Plates were then washed three times in washing buffer (PBS with 0.01% Tween 20) and blocked for 2 h at room temperature (RT) with blocking buffer [20 mM Tris, (pH 8.0); 150 mM NaCl; 0.01% Tween 20; 2% FCS; 0.1% BSA]. Serum samples diluted in blocking buffer were then added and incubated for 1 h at RT, washed three times with washing buffer and appropriate horseradish peroxidase-conjugated species-specific secondary antibody diluted in blocking buffer added, and incubated for 1 h at RT. Plates were then washed three times, and the bound antibodies were detected by colorimetry. Lethal toxin-neutralizing antibody titers were determined essentially as described previously using J774.1 cells (15). Anti-vaccinia–neutralizing antibody titers were determined by a plaque reduction assay as previously reported (11).

Animals, Immunizations, and B. anthracis Spore Challenges.

Female New Zealand White rabbits ≈10 wk of age were purchased from Myrtle's Rabbitry. Female A/J mice 6–8 wk of age were purchased from the National Cancer Institute's animal resources program. Rabbits were immunized twice, 4 wk apart, s.c., with the respective vaccinia virus recombinants at a dose of 1 × 10⁷ pfu in a volume of 0.5 mL Biothrax/AVA vaccine. This was administered intramuscularly with the same schedule as for vaccinia recombinants; the dose consisted of 50 μL of Biothrax/AVA vaccine in 450 μL of PBS. Rabbits were challenged with a mean dose of 262 LD₅₀ of B. anthracis (Ames strain) obtained from BEI Resources by oro-nasal inhalation under appropriate biosafety containment at the Southern Research Institute, Birmingham, AL. In immunizing A/J mice, the vaccinia recombinant viruses were administered s.c. at a dose of 1 × 10⁷ pfu in a 100-μL volume. Biothrax/AVA vaccine was administered s.c. in a 100-μL volume after 25 μL of Biothrax/AVA vaccine was diluted in 75 μL of PBS. Challenging of mice with the spores of toxigenic acapsular B. anthracis strain 7702 (pXO1+; pXO2−) was done as described previously (24) using a six-jet Collision nebulizer with nose only exposure system to deliver a 20-LD₅₀ dose of spores. Housing and care of animals were carried out in accordance with the American Association for Accreditation of Laboratory Animal Care standards in accredited facilities. The experimental design of this study was approved by the Institutional Animal Care and Use Committees.

Statistical Analyses.

Statistical analysis was by unpaired Student's t test, one-way ANOVA with pairwise comparisons, and two-way ANOVA with pairwise comparisons. P < 0.05 was considered significant.