Comparison of homologous and heterologous vaccination strategies for combating disease caused by Burkholderia pseudomallei

Abstract

Introduction: Melioidosis is a major cause of disease and mortality in endemic tropical regions, and the etiologic agent, Burkholderia pseudomallei, is being isolated increasingly from an expanded range of environmental and clinical sources in locations including the United States. The disease can have multi-faceted clinical presentations and requires a complex and protracted treatment regimen which is confounded by resistance of this microbe to numerous antibiotics. Thus, prophylactic countermeasures are needed; however, a vaccine has yet to be licensed for human use. Since B. pseudomallei is classified as a Tier 1 select agent, the development of a safe and effective vaccine is both a military and public health need. Our laboratories have focused on the development of vaccines composed of live attenuated strains and defined subunit antigens.

Methods: In the current study, we evaluated homologous and heterologous combinations of candidate subunits and live vaccines in a murine aerosol model of melioidosis to determine the effects of vaccine composition and delivery scheme on protection in conjunction with immune responses and bacterial clearance.

Results: Both strategies provided significant protection against lethal aerosol challenges, and the accumulated data support that a heterologous vaccination strategy employing capsular polysaccharide conjugate and Hcp1 subunits and a live but highly safe capsular polysaccharide-producing surrogate strain of B. thailandensis is an effective and potentially agile prophylactic strategy.

Keywords: Burkholderia pseudomallei; heterologous; immunity; melioidosis; mice; protection; vaccines.

Figure 1

(A) Overview of the immunization and challenge strategy for the assessment of live attenuated, subunit and homologous vaccines. Some vaccinated animals were given the live attenuated and subunit vaccines at the same time but at distinct sites (not in the same syringe and the ΔilvI live attenuated strain was delivered in a distinct site than the protein subunit vaccine). The subunit vaccines were delivered with the adjuvant Alhydrogel. The quantities of vaccine components are indicated in the box, and the vaccines were administered twice by the subcutaneous (sc) route, on days 0 and 28. The mice were exposed to B. pseudomallei K96243 (4 LD₅₀) by the aerosol route day 63. Created in BioRender. Biryukov, S. (2025) https://BioRender.com/otr69lv. (B) Survival of homologously vaccinated C57BL/6 mice challenged with whole body aerosol challenges of B. pseudomallei K96243 (4 LD₅₀, ~1,630 CFU). The mice (n = 10/group) were vaccinated with E555 ΔilvI (purple), Hcp1 + CRM197-CPS + Alhydrogel (black), Hcp1 + CRM197-CPS + Alhydrogel + E555 ΔilvI (green) or CRM197-CPS + Alhydrogel + E555 ΔilvI (red), as indicated in Figure 1A . Control animals received two doses of PBS (blue). The mice were monitored for 60 days and those that succumbed, or were euthanized, were recorded daily. ^*The day 21 survival rates of three vaccine groups were greater than that of the PBS controls (p = 0.039). ^**The day 60 survival rate of E555 ΔilvI -vaccinated mice was greater than that of the PBS control group (p = 0.035).

Figure 2

The quantitation of bacterial CFU from tissues of C57BL/6 mice infected with B. pseudomallei K96243. The numbers of CFU/g organ were assessed following tissue homogenization, serial dilution and spreading aliquots onto agar medium three days after challenge for lungs (A) and spleens (B). The data points represent colony counts for each of six animals per group. Bacterial burdens in the lungs (C) and spleen (D) of vaccinated mice that survived to the end of study are shown. The horizontal lines are the geometric means.

Figure 3

(A) Overview of the immunization and challenge strategy for the assessment of Burkholderia vaccine candidates delivered via homologous and heterologous vaccination strategies. The vaccines were administered twice by sc route, on days 0 and 28, and the mice were exposed to B. pseudomallei K96243 (6.0 LD₅₀ to groups 1–3 and 3.4 LD₅₀ to groups 4 - 8) by the aerosol route four weeks later; n = 15 mice/group. As detailed in Table 3 , groups 2 and 3 received homologous vaccines for the prime and boost doses; groups 5–8 received the heterologous vaccines; and groups 1 and 4 included the PBS controls. The subunit vaccines were delivered with the adjuvant Alhydrogel and immunostimulant CpG 2006 (10 µg). Created in BioRender. Biryukov, S. (2025) https://BioRender.com/oji8j26. (B) Survival curves of vaccinated C57BL/6 mice challenged with whole body aerosol challenges of B. pseudomallei K96243 (6.0 LD₅₀, ~2,400 CFU). The mice (n = 10/group) received a prime and boost with the same vaccine (homologous), as shown in Table 3 . The subunit vaccines included Alhydrogel and the CpG immunostimulant. Control animals received two doses of PBS. The mice were monitored for 60 days and those that succumbed to infection, or were euthanized, were recorded daily. (C) Survival curves of vaccinated C57BL/6 mice challenged with whole body aerosol challenges of B. pseudomallei K96243 (3.4 LD₅₀, ~1,360 CFU). The mice (n = 10/group) received heterologous vaccinations in which the prime and boost were different vaccines (separated by “/” in the legend), as shown in Table 3 . The subunit vaccines included Alhydrogel and CpG. Control animals received two doses of PBS. The mice were monitored for 60 days and those that succumbed to infection, or were euthanized, were recorded daily.

Figure 4

IFNγ-secreting splenocytes obtained from control and vaccinated C57BL/6 mice twenty-seven days post boost following stimulation with 10 µg/ml Hcp1. The splenocyte response of mice (n = 5/group) was assessed as spot forming cells (SFC), adjusted to 10⁶ cells per well. Values represent the GM ± the GSE.

Figure 5

(A) Overview of the immunization and challenge strategy for the assessment of Burkholderia vaccine candidates delivered via homologous and heterologous vaccination strategies. The vaccines were administered twice by sc route, on days 0 and 28; n = 18 mice/group. Mice were exposed to B. pseudomallei K96243 (3.4 LD₅₀) by the aerosol route four weeks later; n = 13 mice/group. As detailed in Table 3 , groups 2 and 3 received homologous vaccines for the prime and boost doses; groups 7, 8, and 9 received the heterologous vaccines; and group 1 included the PBS controls. The subunit vaccines were delivered with the adjuvant Alhydrogel and, except for group 9, the immunostimulant CpG. Created in BioRender. Biryukov, S. (2025) https://BioRender.com/rfgku3j. (B) Survival of vaccinated C57BL/6 mice challenged with whole body aerosol challenges of B. pseudomallei K96243 (3.4 LD₅₀s, ~1,610 CFU). The heterologous and homologous vaccine groups (n = 10 each) are listed in Table 3 . The subunit vaccines included Alhydrogel and CpG, except group 9 as shown in Table 3 . The mice were monitored for 60 days and those that succumbed to infection, or were euthanized, were recorded daily. For heterologous vaccine groups 7, 8, and 9 the different prime and boost formulations are separated by “/” in the legend.

Figure 6

Bacterial burdens in the blood and organs of vaccinated mice infected with B. pseudomallei K96243. The numbers of CFU/g organ were assessed following tissue homogenization, serial dilution and spreading aliquots onto agar medium three days after challenge for lungs (A) and spleens (B). The data points represent colony counts for each of three animals per group. Bacterial burdens in the lungs (C) and spleens (D) of vaccinated mice that survived to the end of study are shown, all spleens were negative for B. pseudomallei. None of the PBS control mice survived. The horizontal lines are the GM. One mouse was found dead the day of necropsy and was shown to be positive for B. pseudomallei in both lungs and spleen [shown as red data points in panels (C, D)] but was omitted from the GM calculation because of the time between death and sample collection was undetermined.

Figure 7

IFNγ-secreting splenocytes obtained from control and vaccinated C57BL/6 mice twenty-seven days post boost following stimulation with 10 µg/ml Hcp1. The splenocyte response of mice (n = 5/group) was assessed as spot forming cells (SFC), adjusted to 10⁶ cells per well. Values represent the GM ± the GSE.

Figure 8

The combined survival data of mice from two experiments. The survival results of groups 7, 8, and the PBS controls, obtained from the studies depicted in Figures 4 , 6 , are shown (n = 20/group). All mice had been challenged with the same whole body aerosol dose of B. pseudomallei K96243 (3.4 LD₅₀s).