Development of a Nanoemulsion-Based Burkholderia cenocepacia Mucosal Vaccine as Prophylaxis for Pan-resistant Bacterial Infection Associated with Cystic Fibrosis and Chronic Lung Infection
The Carroll Haas Foundation and The Wheeler Family Foundation
Paul Makidon, DVM, PhD, Primary Investigator, Research Investigator, Department of Internal Medicine and MNIMBS
James R. Baker Jr., MD, Principal Investigator, Professor of Internal Medicine, Director, MNIMBS
John Lipuma, MD, Professor of Epidemiology, School of Public Health
Anna U. Bielinska, PhD, Research Assistant Professor, Department of Internal Medicine and MNIMBS
Burkholderia cepacia complex (Bcc) are opportunistic bacteria associated with life threatening illness in persons with cystic fibrosis and chronic lung infection. Once Bcc colonization is established, these antimicrobial-resistant and biofilm-forming bacteria are difficult to eradicate and are associated with increased rates of morbidity and mortality. At present, no vaccines are available to prevent the Bcc infection. There is currently a paucity of published information regarding the development of vaccines designed to prevent Burkholderia colonization. This work expands on the recent studies published by Bertot et al 2007 where successful protective immune responses were generated in mice using a B. multivorans OMP-based vaccine. Here we evaluate an experimental mucosal vaccine against Bcc using a novel mucosal adjuvant (nanoemulsion) and a novel B. cenocepacia-based OMP antigen. The OMP antigen derived from B. cenocepacia was mixed with either nanoemulsion or with PBS and delivered intranasally to CD-1 mice. Serum analysis showed robust IgG and mucosal secretory IgA immune responses in vaccinated versus control mice. The antibodies had cross-neutralizing activity against both B. cenocepacia and B. multivorans species. We found that immunized mice were protected against pulmonary colonization with B. cenocepacia and have also identified a new immunodominant epitope, a 17kD OmpA-like protein which is highly conserved between Burkholderia and Ralstonia species.
Specific Aim 1: To characterize the immunogenic potential after nasal application of a mixture of an outer membrane protein (OMP) derived from B. cenocepacia and nanoemulsion (OMP-NE).
Specific Aim 2: To evaluate the ability of sera from mice vaccinated with OMP-NE to neutralize B. cenocepacia and B. multivorans, and test for protective immunity following experimental B. cenocepacia pulmonary infection.
Studies and Results
Identification of immunoreactive proteins
To identify immunoreactive proteins in OMPs from B. cenocepacia, mice were intranasally immunized and boosted 4 weeks following prime vaccination with 15 μg OMP in 20% NE or 15 μg OMP in PBS. Serum from individual mice were used to probe electrophoretically separated and transferred OMP protein fractions. Immunoreactive OMP proteins were identified via silver stain (Fig. 1A) and western blot (Fig. 1B and Fig. 1C) Major reactive bands were documented at 62, 45, 17, and 10 kDa in both cases; however, the intensity of the bands was much higher with serum from mice immunized with the NE-based vaccine (Fig. 1B) as compared to the blot probed with an equivalent serum dilution from mice immunized with the OMP in PBS preparation (Fig. 1C).
Fig. 1. Characterization of the OMP preparation. Volumetrically-loaded silver stain A) and western blot of OMP preparation (B-C). Lane A. Protein from the supernatant produced after the 6000 x g centrifugation was loaded; Lane B. An equal volume of the supernatant from the 100,000 x g centrifugation; Lane C. Crude OMP (the re-suspended pellet fraction of the 100,000 x g spin); Lanes D-J. Endotoxin-depleted OMP fractions (the successive flow-through portions of the endotoxin column); Lanes K-N. Endotoxin column retentant fractions (the successive fluid regenerated from the column after the addition of sodium deoxycholate). B) Western blot probed with serum from and individual mouse immunized with the endotoxin-depleted OMP-NE preparation. C) Western blot probed with serum from mice immunized with the OMP in PBS preparation.
The band with the highest intensity was located at ~ 17 kDa (Fig. 1B). For the purpose of identification, the 17 kDa band was isolated from the gel and identified by MALDI-TOF analysis. The protein was identified as Burkholderia cepacia outer membrane lipoprotein A (OmpA) with a MW of 16.396 kDa (Ortega et al., 2005; Plesa et al., 2004). Sequence analysis using National Center for Biotechnology Information (NCBI) protein BLAST confirmed that these proteins are present in other closely related Burkholderia species. A high degree of protein homology (87.9% sequence homology) was identified in numerous strains of Burkholderia and Ralstonia organisms and highly conserved amino acids from the OmpA family residues are present in the 17 kDa protein sequence.
Intranasal immunization with OMP-NE induces anti-OMP specific antibodies
The humoral immune responses against the OMP induced by nasal vaccine with OMP were characterized in vivo in the CD-1 mice. Intranasal vaccination with either 5 μg or 15 μg OMP preparations + NE (OMP-NE) resulted in high serum titers of OMP-specific IgG antibodies of 2.8 x 105 and 5.1 x 105 at 6 weeks, respectively, following primary vaccination (Fig. 2A). OMPs without adjuvant were immunogenic and resulted in serum anti-OMP IgG titers of 1.9 x 104 and 3.8 x 104 six weeks following primary i.n. immunization. However, treatment groups with NE as an adjuvant responded significantly higher (13 to 30 fold) than groups without adjuvant at all time points following the boost (p< 0.05). Further, mice immunized with OMP in PBS did not demonstrate a significant boost effect following the second vaccination (Fig. 2A).
To further characterize the OMP-NE vaccine, we evaluated its ability to elicit specific antibody production in bronchiolar secretions. Mucosal antibody production may be important for protection against Burkholderia colonization since secretory antibodies are thought to be critical effectors in protection against mucosal respiratory pathogens (Nelson et al., 1993). Mucosal immune responses were evaluated in bronchoalveolar lavage (BAL) fluid of animals immunized with 5 μg OMP-NE or 5 μg OMP in PBS. Comparable levels of anti-OMP antibodies in BALs were detected in mice immunized either with OMP-NE or OMP without NE adjuvant (Fig. 2B).
Fig. 2. Antibody responses against B. cenocepacia OMP. A) ELISA results of the IgG response in serum post-immunization with the OMP preparation with or without nanoemulsion. Mice (n=10/group) received a primary vaccination and a boost 4 weeks following prime. Serum anti-OMP IgG antibody concentrations are presented as mean of endpoint titers in individual sera ± SEM. * indicates a statistical difference (p<0.05) in the anti-OMP IgG titers. B) Mucosal antibodies sIgA and IgG against the OMP after nasal vaccine with 5 μg OMP ± NE. sIgA and IgG were measured in BAL solution. The OD levels were normalized to total protein content within the samples. BAL were collected 2 weeks following boost vaccination administered at 4 weeks.
OMP-NE immunization yields a balanced Th1/Th2 cellular response
The analysis of the serum IgG subclass was performed to determine the T helper-type bias of the cellular response. The pattern of IgG subclass distribution (illustrated in titer’s ratio) shows that i.n. immunization with OMP-NE resulted in skewing of Th1-type antibodies (IgG2b) versus Th2-type IgG1 subclass antibodies (p =0.048) (Fig. 3A). In comparison, immunization with OMP in PBS produced similar levels of IgG1 and IgG2b antibodies (Fig. 3A).
OMP-specific cellular responses were characterized in splenocytes obtained 6 weeks following primary vaccination with OMP-NE. The cells were stimulated with endotoxin-depleted OMP and then evaluated for specific cytokine production. In splenocytes collected from OMP-NE vaccinated mice, Th1 cytokines IFN-γ and IL-2 production increased 22.1 and 18.6-fold respectively over that of splenocytes from non-vaccinated mice (p = 0.01 and 0.003, respectively) (Fig. 3B). IFN-γ was equally induced in splenocytes from mice immunized with OMP in PBS. The Th2 cytokines IL-4, IL-5, IL-6, and IL-10 were minimally induced (≤ 5.22-fold increase) in both OMP-NE and OMP immunized mice in equivalent amounts. The pattern of splenic cytokine expression and the IgG subclass distribution results suggested a balanced Th1/Th2 response to OMP-NE vaccination.
Fig. 3. Type of cellular immune responses induced by nasal OMP-NE vaccine. A) Serum from mice immunized with 5 μg OMP mixed with either NE (OMP-NE) or with PBS (OMP-PBS) was analyzed for antibody subtype distribution. Mice (n=10/group) received a prime vaccination and a boost 4 weeks following prime. The analysis was performed 2 weeks following booster vaccination administered at 4 weeks. The results are presented as ratio of the specific subclass IgG to the overall IgG titer. *: indicates statistical difference (p<0.05) between IgG2b and IgG1 subtypes. The error bars represent the value of the standard error distribution. B) Cytokine profiling of splenocytes of mice immunized with 5 μg. mixed with either NE (OMP-NE) or with PBS (OMP-PBS). The spleens were harvested 2 weeks following booster vaccination administered at 4 weeks. Data is represented as fold change ± SEM comparing OMP-activated versus non activated splenocytes and is normalized to responses in non-vaccinated mice. *: indicates statistical difference (p<0.05) between OMP-NE and OMP in PBS groups.
Intranasal vaccination with OMP-NE results in cross-protective neutralizing serum antibody
B. cenocepacia neutralizing antibodies were determined using a colony reduction assay. The mice intranasally vaccinated with 5 μg or 15 μg OMP-NE produced serum-neutralizing antibodies that inhibited 92.5% or 98.3% of B. cenocepacia colonies, respectively, as compared to serum from non-vaccinated mice. The mice vaccinated with OMP lacking adjuvant also demonstrated a relatively high level of inhibition, resulting in 33.3% and 46.7% (for 5 μg and 15 μg OMP-PBS, respectively) cfu reduction. The statistical analysis showed that B. cenocepacia growth was significantly inhibited by serum derived from OMP-NE as compared to serum from those who were immunized with the OMP preparation alone.
To evaluate if the OMP-NE can produce cross-reactive protection, serum from mice vaccinated with 15 μg OMP-NE was also incubated with B. multivorans. The B. multivorans was selected because it is the most common isolate cultured from CF patients infected with Bcc organisms (Baldwin et al., 2008). The serum inhibited B. multivorans growth by 80.1% and by 49.8% derived from mice vaccinated with OMP-NE and OMP without adjuvant, respectively, suggesting significant cross-protective antibodies following immunization with either OMP-NE or OMP in PBS.
Immunization with OMP-NE protects against pulmonary B. cenocepacia challenge and reduces incidence of sepsis
The protective effect of intranasal immunization was further tested in vivo in a lung infection model. The clearance of B. cenocepacia from pulmonary tissue was evaluated 6 days following intra-tracheal inoculation in mice that were intranasally immunized with the OMP-NE or the OMP without an adjuvant. Vaccination with 15 μg OMP-NE resulted in significantly higher rates of pulmonary clearance (p = 9.2x10-3) as compared to the non-vaccinated mice (Fig. 4A). At day 6, the average pulmonary bacterial load was 22.5 ± 26.2 cfu in mice vaccinated with 15 μg OMP-NE, compared to 1.28 x 106 ± 3.36 x 106 cfu per lung in non-vaccinated mice, representing a greater than 5-log reduction in the bacterial load.
Fig 4. Pulmonary and splenic colonization assay. Mice (n=10/group) received a primary vaccination and a boost 4 weeks following prime with either 5 μg or 15 μg OMP ± 20% NE. Colonization studies were performed 6 weeks following boost vaccination administered at 4 weeks. A) Pulmonary tissue associated and B) Splenic tissue associated cfu determined at six days following intratracheal challenge of 5 x 107 cfu of B. cenocepacia. Data is represented as CFU/organ ± SEM.
Splenic colonization following pulmonary inoculation with B. cenocepacia was evaluated as a means of assessing sepsis (Fig. 4B). Vaccination with 15 μg OMP-NE significantly reduced the incidence of bacteria in the spleens 6 days following the pulmonary
challenge of individual mice from an average of 3.54 x 103 ± 6.97 x 103 cfu per spleen in non-vaccinated mice to 2.5 ± 5 cfu per spleen in vaccinates (p = 0.0307).
Our study demonstrates protective immune responses generated against B. cenocepacia following mucosal vaccination with OMP-NE. OMP-NE formulations induces robust, and specific humoral and cellular responses, resulting in protective immunity. The 17 kDa OmpA-like protein shows potential for future vaccine development and our study provides a rational basis for vaccines using recombinant OMP protein mixed with NE as an adjuvant.
Makidon PE, Knowlton J, Groom JV II, Blanco LP, Lipuma JJ, Bielinska AU, Baker JR Jr. Induction of immune response to the 17 kDa OMPA Burkholderia cenocepacia polypeptide and protection against pulmonary infection in mice after nasal vaccination with an OMP nanoemulsion-based vaccine. Med Microbiol Immunol 199(2), 81-92, 2010.