Role of Human Opsonins in Killing Salmonella enterica Serovar Typhi

INFECTION AND IMMUNITY, Aug. 2011, p. 3188–3194 0019-9567/11/$12.00 doi:10.1128/IAI.05081-11 Copyright © 2011, American Society for Microbiology. All Rights Reserved.

Vol. 79, No. 8

Antibodies in Action: Role of Human Opsonins in Killing Salmonella enterica Serovar Typhi䌤 Janet C. Lindow,1* Kelly A. Fimlaid,1 Janice Y. Bunn,2 and Beth D. Kirkpatrick1 University of Vermont College of Medicine, Department of Medicine, Vaccine Testing Center and Unit of Infectious Diseases, Burlington, Vermont,1 and University of Vermont College of Engineering and Mathematical Sciences, Burlington, Vermont2 Received 16 March 2011/Returned for modification 4 April 2011/Accepted 19 May 2011

Although vaccines have been available for over a century, a correlate of protection for typhoid fever has yet to be identified. Antibodies are produced in response to typhoid infection and vaccination and are generally used as the gold standard for determining vaccine immunogenicity, even though their role in clearance of Salmonella enterica serovar Typhi infections is poorly defined. Here, we describe the first functional characterization of S. Typhi-specific antibodies following vaccination with a new vaccine, M01ZH09 (Ty2 ⌬aroC ⌬ssaV). We determined that postvaccination sera increased the uptake of wild-type S. Typhi by human macrophages up to 2.3-fold relative to prevaccination (day 0) or placebo samples. These results were recapitulated using immunoglobulins purified from postvaccination serum, demonstrating that antibodies were largely responsible for increases in uptake. Imaging verified that macrophages internalized 2- to 9.5-fold more S. Typhi when the bacteria were opsonized with postvaccination sera than when the bacteria were opsonized with day 0 or placebo sera. Once inside macrophages, the survival of S. Typhi was reduced as much as 50% when opsonized with postvaccination sera relative to day 0 or placebo serum samples. Lastly, bactericidal assays indicated that antibodies generated postvaccination were recognized by complement factors and assisted in killing S. Typhi: mean postvaccination bactericidal antibody titers were higher at all time points than placebo and day 0 titers. These data clearly demonstrate that there are at least two mechanisms by which antibodies facilitate killing of S. Typhi. Future work could lead to improved immunogenicity tests associated with vaccine efficacy and the identification of correlates of protection against typhoid fever. both (13, 14). Several next-generation vaccines, designed to optimize efficacy and simplify delivery, are currently in human trials, including a single-oral-dose typhoid vaccine, M01ZH09 (S. Typhi Ty2 ⌬aroC ⌬ssaV) (4, 14, 17, 22, 28, 31, 32, 41). Human immune responses to typhoid infections involve innate and antigen-specific humoral and cellular immune responses; however, the relative contribution of each arm of the immune system is not well understood. Work performed by several groups over the past decade has clarified the role of cell-mediated immune responses to S. Typhi, particularly the role of CD8⫹ T cells (15, 30, 40). The role of the humoral immune response is not as well defined. Many large-scale field trials have demonstrated that S. Typhi-specific antibodies are produced in a majority of subjects following vaccination or natural illness, but the function or mechanism of protection provided by S. Typhi-specific antibodies is currently uncharacterized (8, 14, 23, 24, 31, 32, 39, 46). A better understanding of the function of antibodies mounted in response to disease or vaccination addresses major challenges in understanding humoral immune responses to typhoid disease and aids in the evaluation of new typhoid vaccines. This work capitalizes on clinical specimens following study of the candidate typhoid vaccine M01ZH09 (S. Typhi Ty2 ⌬aroC ⌬ssaV, here called ZH9), a single-oraldose live attenuated vaccine with two independently attenuating mutations in healthy American adults (21, 22, 31). We describe the functional role of opsonins generated following vaccination with ZH9 in the uptake and killing of wild-type S. Typhi.

Typhoid fever, a food- and waterborne disease, results in an estimated 21 million illnesses and 200,000 deaths annually (6). The greatest disease burden is borne by people living in resource-poor regions of the world who lack access to clean drinking water. Salmonella enterica serovar Typhi, a humanrestricted, intracellular, Gram-negative bacterium, is the causative agent of typhoid fever. During an infection, bacteria cross the intestinal epithelial barrier to invade phagocytic cells in the lamina propria, allowing them to quickly spread via the bloodstream to reticuloendothelial organs, such as the liver and bone marrow (35, 49). Antibiotic resistance in S. Typhi isolates has risen dramatically since the 1980s, which intensifies the need for new publichealth-based strategies, prudent use of antibiotics, and nextgeneration vaccines (1, 2, 37). Although typhoid fever vaccines have been available for over a century, they have ranged greatly in efficacy and reactogenicity (13, 14). There are currently two safe and effective vaccines, Ty21a and Vi polysaccharide (Vi) vaccines, licensed in 56 and 92 countries, respectively (13, 14, 23, 24, 49). However, both vaccines have drawbacks that necessitate the development of next-generation typhoid vaccines: Ty21a requires 3 or 4 oral doses, while Vi requires a needle injection, and refrigeration is necessary for

* Corresponding author. Mailing address: Unit of Infectious Diseases and Vaccine Testing Center, University of Vermont College of Medicine, 95 Carrigan Drive, Stafford Hall 110, Burlington, VT 05405. Phone: (802) 656-7717. Fax: (802) 656-0881. E-mail: janet.lindow @uvm.edu. 䌤 Published ahead of print on 31 May 2011. 3188

VOL. 79, 2011

POSTVACCINATION ANTIBODIES KILL SALMONELLA TYPHI MATERIALS AND METHODS

Cell culture and bacterial strains. THP-1 monocytes (catalog number TIB202) and Salmonella enterica serovar Typhi wild-type strain Ty2 (catalog number 19430) were purchased from the American Type Culture Collection (Rockville, MD) and maintained using standard methods. Briefly, THP-1 was grown in RPMI 1640 (Gibco A10491) supplemented with 10% fetal bovine serum and 50 ␮M ␤-mercaptoethanol with or without 100 U penicillin/100 ␮g/ml streptomycin at 37°C, 5% CO2. Ty2 was grown in Luria broth (LB) overnight (O/N) (16 to 24 h) with vigorous shaking at 37°C. BK26 (Ty2/pJL1) was grown identically to Ty2, except that 100 ␮g/ml ampicillin was added to LB to select for the plasmid pJL1. Reagents. RPMI 1640 (A10491), goat anti-human IgG-horseradish peroxidase (HRP), goat anti-human IgA-HRP, and fetal bovine serum were purchased from Gibco Invitrogen (Carlsbad, CA); phorbol 12-myristate 13-acetate (PMA), ␤-mercaptoethanol, and cytochalasin D (Cyto D) from Sigma-Aldrich, Inc. (St. Louis, MO); 4⬘,6-diamidino-2-phenylindole (DAPI) from Invitrogen (Carlsbad, CA); donkey polyclonal antibodies to Salmonella conjugated to fluorescein isothiocyanate (FITC) from KPL (Gaithersburg, MD); and a Vi antigen agglutination test from BD (San Jose, CA); all other reagents were purchased through Fisher unless otherwise specified. Strain construction. All strains were constructed using standard methods. Briefly, pJL1 was transformed into S. Typhi Ty2 (BK7), selecting for ampicillin resistance, creating strain BK26. pJL1 construction. mCherry (a fluorescent red protein gene) was amplified using Taq Master Mix and oJCL5 (5⬘-ATCTAGAAAGAAGGAGATATACAT GGTTTCCAAGGGC) and oJCL2 (5⬘-ATAAAGCTTTTATTATTTGTACAG CTC) from pAWY3-mCherry (a gift from M. Berkmen). mCherry was cloned into the XbaI-HindIII sites on pFPV25.1 (a gift from R. Valdivia), replacing gfp and generating pJL1. Ethics statement. All work with human subjects was reviewed and approved by the University of Vermont Committees on Human Research and the Johns Hopkins Medicine Institutional Review Board prior to initiation of clinical studies. Sera from healthy volunteers. Sera were collected from healthy adults in the United States with no prior exposure to S. Typhi who had been vaccinated with the live attenuated S. Typhi M01ZH09 vaccine (Emergent Biosolutions) or given a placebo (31). Sera were separated within 2 h of blood draw in SST vacutainers (BD Biosciences) by centrifugation (10 min; 1,200 relative centrifugal force [RCF]) and stored at ⫺80°C. Volunteers received 7.5 ⫻ 109 (cohort 2) or 1.7 ⫻ 1010 (cohort 4) CFU of vaccine. A total of 10 placebo-injected and 11 vaccinated volunteers were used in this study. Phagocytosis assays. THP-1 monocytes (5 ⫻ 105 per well) were seeded in 24-well plates (Costar) and differentiated with 81 nM PMA for 20 to 24 h, washed twice with phosphate-buffered saline (PBS), and incubated at 37°C, 5% CO2 in 0.5 ml RPMI 1640 medium for 2 to 4 h. Where specified, 2 ␮M cytochalasin D was added to the THP-1 monocytes for 1 h prior to phagocytosis. Ty2 was grown to stationary phase as described above. The cells were positive for Vi antigen by agglutination test. One milliliter of O/N culture was washed once in 1 ml of PBS. Cell density was measured, and the culture was diluted to 2 ⫻ 108 CFU/ml. For an MOI of 20, 50 ␮l bacteria (2 ⫻ 108 cells/ml) was opsonized with 5% heat-inactivated (56°C; 30 min) human serum in PBS for 30 min at 37°C. Opsonized Ty2 bacteria were spun (200 ⫻ g) onto differentiated THP-1 monolayers for 5 min to synchronize phagocytosis. The cultures were incubated for 30 min at 37°C, 5% CO2. Phagocytosis was stopped with 3 ice-cold PBS washes, and 1 ml of RPMI 1640 with 200 ␮g/ml gentamicin was added per well. After 1.5 h at 37°C, 5% CO2, the cells were washed twice with cold PBS, and macrophages were lysed with 250 ␮l 1% Triton-X for 10 min at 37°C. For 48-h time points, macrophages were incubated in 1 ml of RPMI 1640 plus 10 ␮g/ml gentamicin for 46 h at 37°C, 5% CO2 before lysing. The lysed cultures were immediately diluted in PBS and plated for viable bacteria on LB plates at 37°C O/N. The viability of S. Typhi was not affected by the described lysis conditions. S. Typhi alone was not viable after 2 h of incubation in RPMI plus 200 ␮g/ml gentamicin. Phagocytic-index (PI) experiments. THP-1 cells were differentiated O/N on glass coverslips (BellCo Glass) in 24-well plates. Phagocytosis was performed similarly to that described above with the following exceptions. Ty2 constitutively expressing mCherry from a plasmid was used: strain BK26. Following phagocytosis as described above, cells were fixed with 4% paraformaldehyde in PBS for 20 min at room temperature and washed 3 times with PBS, and extracellular bacteria were stained with anti-Salmonella common structural antigens (CSA)– FITC (0.003 mg/ml) for 1 h in the dark. Nuclei and nucleoids were stained with DAPI (50 ␮g/ml). The cells were visualized on a Nikon Eclipse Pi fluorescence microscope using a 100⫻ oil immersion objective. Digital images were captured

3189

using a Q imaging camera and NIS Elements ND2 software. The images were processed using Image J and Adobe Photoshop Elements 7 software. Immunoglobulin purification and phagocytosis assays. IgG, IgA, and IgM were isolated from serum samples (days 0, 7, 14, and 28) using Pierce Thiophilic Adsorbent T-Gel according to the manufacturer’s instructions. Briefly, 100 ␮l of serum was incubated with 250 ␮l of equilibrated T-Gel resin (washed with binding buffer [BB] [0.5 M potassium sulfate plus 50 mM sodium phosphate]) for 1 h at room temperature with shaking. The resin was washed 5 times using 1 ml BB. Immunoglobulins were eluted with 1 ml of elution buffer (50 mM sodium phosphate, pH 8). Fractions were tested for human IgA or IgG heavy chain by Western blot analysis using goat anti-human IgG-HRP (1:250,000) or anti-human IgA-HRP (1:250,000). Phagocytosis assays were performed as described above, except 8 to 14 ␮g of purified immunoglobulins was used to opsonize bacteria instead of 5% serum. The concentration of purified immunoglobulins was selected after titrating purified immunoglobulins from 1 ␮g to 200 ␮g. The normal range for total immunoglobulins in 5% serum is 25 to 90 ␮g. Serum bactericidal antibody assay. Heat-inactivated serum (56°C; 30 min) was serially diluted from 1:250 to 1:16,000 in PBS. Twofold dilutions were mixed with 5% (final concentration) baby rabbit complement (Pel-Freez Biologicals) and 250 CFU of stationary-phase wild-type S. Typhi Ty2. Negative controls contained Ty2 only and Ty2 plus complement. To ensure serum did not contain any complement after heat inactivation, Ty2 plus serum (no exogenous complement) was tested for bactericidal activity for a few samples. Mixes were incubated for 45 min at 37°C, plated on LB agar plates, and incubated O/N at 37°C. The bactericidal titer is defined as the inverse of the dilution of serum at which ⱕ50% of bacteria survived relative to the bacteria-plus-complement control. All samples were assayed in duplicate, and the average of the results is reported. Statistical analysis. A repeated-measures analysis of variance was used to examine bactericidal antibody titers, replication, or uptake with vaccine (active vaccine versus placebo) as the grouping factor and time as the repeating factor. Phagocytic-index data were examined using a single-group repeated-measures analysis, with time as the repeating factor. Data not meeting distributional assumptions were transformed into ranks prior to analysis. All data were normalized to prevaccination samples. An unpaired two-tailed t test (95% confidence interval [CI]) was used to compare Cyto D-treated and untreated samples. Repeated-measures analyses were performed using SAS version 9.2; t tests were computed using PRISM 5.0.

RESULTS Postvaccination opsonins increase uptake of wild-type S. Typhi by human macrophages. Opsonins bind to many types of pathogens and facilitate their uptake by phagocytic cells (i.e., macrophages) via phagocytic cell receptors. Using human macrophage-like cells, we used an opsonophagocytosis assay to determine differences in phagocytic uptake of wild-type S. Typhi opsonized with pre- or postvaccination serum. PMA-differentiated THP-1 monocytes were incubated with wild-type S. Typhi, opsonized with 5% serum (prevaccination [day 0] and 7 days [day 7], 14 days [day 14], or 28 days [day 28] postvaccination), and the number of bacteria phagocytosed after 2 h was determined for each sample. The fold increase in phagocytosis was calculated by dividing the number of bacteria phagocytosed when opsonized with postvaccination serum by the number when incubated with autologous day 0 serum (the prevaccination time point) for each sample. Significantly more bacteria were taken up by macrophages when opsonized with postvaccination sera relative to prevaccination (day 0) or placebo control sera (P ⬍ 0.05) (Fig. 1A). No differences in phagocytosis were seen for the placebo samples relative to their autologous day 0 samples at any time points (day 7, day 14, and day 28), while all postvaccination time points in vaccinees showed significantly increased phagocytosis relative to day 0 (P ⬍ 0.001). The uptake across days differed significantly for day 7 relative to day 28 (P ⬍ 0.05), while days 7 and 14 and days 14 and 28 did not differ significantly from one another. Overall, S.

3190

LINDOW ET AL.

FIG. 1. Opsonization with postvaccination serum increases uptake of wild-type S. Typhi by human macrophages. (A) Stationary-phase wild-type (wt) Ty2 S. Typhi bacteria were untreated (stippled bar) or opsonized with 5% heat-treated serum from ZH9 vaccinees (black bars) or placebo controls (white bars) and incubated with THP-1 macrophages at a multiplicity of infection (MOI) of 20. All values are the means and standard errors (SE) of 9 vaccinees or 7 placebos tested in duplicate. The values were generated by dividing the number of bacteria phagocytosed at a non-day 0 time point by the autologous day 0 (d0) sample. A significant difference in uptake between the vaccine and placebo groups was observed (P ⬍ 0.05). (B) Differentiated THP-1 macrophages were untreated (white bar) or treated with Cyto D, an actin polymerization inhibitor (black bar), before unopsonized wt S. Typhi (MOI ⫽ 20) was added. CFU/ml is the number of bacteria phagocytosed per ml. *, P ⱕ 0.001 for the difference between a postvaccination time point and day 0; **, P ⬍ 0.01 for phagocytosis of S. Typhi with or without Cyto D.

Typhi-specific opsonins present in immune sera were detectable for at least 4 weeks following vaccination with ZH9 and increased the number of wild-type S. Typhi phagocytosed by macrophages. Interestingly, phagocytosis of unopsonized bacteria was reduced relative to uptake of bacteria opsonized with prevaccination serum. These data suggest that, in addition to the S. Typhi-specific increases, other serum factors also nonspecifically increase opsonization. S. Typhi adheres to host cells via pili, fimbriae, or other surface molecules prior to invasion. To ensure that the effects observed were due to phagocytic uptake of the bacteria rather than adherence of S. Typhi to the macrophages, we used Cyto D to inhibit phagocytosis. Cyto D inhibits actin remodeling within macrophages, a required step in the phagocytosis of bacteria (33). Phagocytosis was reduced more than 2 log units in the presence of Cyto D (Fig. 1B). These results indicate that

INFECT. IMMUN.

FIG. 2. The phagocytic index is increased for postvaccination time points. Shown are micrographs of phagocytosed S. Typhi (red) opsonized with prevaccination (A) or day 7 (B) immune serum at an MOI of 20. Extracellular S. Typhi was stained with FITC (green). (C) Phagocytic indices (average number of bacteria/phagocytosing macrophage) for sera from 4 vaccinees and 1 placebo. The differences in phagocytic indices between postvaccination time points and day 0 were significant: P ⬍ 0.05 for day 7, P ⬍ 0.005 for day 14, and P ⬍ 0.05 for day 28. *, a minimum of 100 macrophages were scored per time point except for sample 2; **, day 28 was not analyzed for subject 4.

the bacteria were internalized by macrophages and did not simply adhere to the cell surface. Postvaccination opsonins increase the phagocytic index. We next visualized the phagocytosis of opsonized S. Typhi in order to determine whether the number of bacteria per macrophage was altered. We constructed a wild-type S. Typhi strain that constitutively expresses mCherry (a fluorescent red protein) from a plasmid. The mCherry S. Typhi strain (BK26) behaves nearly identically to the wild type, as determined by growth in culture and on plates, Vi agglutination tests (both strains are Vi⫹), microscopic visualization of individual bacteria, and opsonophagocytosis assays assessing uptake of bacteria by macrophages (data not shown). In order to distinguish extracellular or adhered bacteria from phagocytosed bacteria, we added an anti-Salmonella antibody conjugated to FITC (green) following phagocytosis, which labeled only extracellular bacteria; macrophages were not permeabilized, and intracellular bacteria were therefore inaccessible to antibody. Macrophages were visualized with DAPI-stained nuclei (Fig. 2A and B). The average number of intracellular bacteria per phagocytosing macrophage (phagocytic index) was then determined for preand postvaccination time points. No major differences in phagocytic indices were noted in the placebo control at any time point: day 0, day 7, day 14, or day 28 (Fig. 2C). However, the phagocytic index was significantly greater for bacteria op-

VOL. 79, 2011

POSTVACCINATION ANTIBODIES KILL SALMONELLA TYPHI

FIG. 3. Antibodies mediate increases in phagocytic uptake of S. Typhi by macrophages. Stationary-phase wt Ty2 S. Typhi was opsonized with ⬃11 ␮g of purified immunoglobulins (white bars) or 5% serum (black bars) from ZH9 vaccinees or a placebo control and incubated with THP-1 macrophages at an MOI of 20. The histograms represent the differences between the values for vaccinee or placebo samples divided by those for their autologous prevaccination samples (gray bar). n ⫽ 3 vaccinees or 1 placebo. Samples were run in duplicate. No difference in uptake at any time point for any sample was observed between purified immunoglobulins and serum groups (P ⫽ 0.19). The error bars indicate SE.

sonized with serum from any of the three postvaccination time points (day 7, day 14, or day 28) than for bacteria opsonized with serum from the autologous day 0 sample for the vaccinees examined, ranging from increases of 2.9- to 9.4-fold (P ⬍ 0.005). These data show that opsonization with postvaccination serum increases the number of bacteria phagocytosed by macrophages and are consistent with the data shown in Fig. 1A. Antibodies are the primary opsonins responsible for increased phagocytic uptake. We suspected that S. Typhi-specific antibodies (IgG, IgA, and IgM at early time points) were the immune opsonins primarily responsible for the observed increases in phagocytosis of S. Typhi. Previous studies showed that all subjects tested in the opsonophagocytosis assays produced IgG and IgA specific to S. Typhi lipopolysaccharide (LPS) following vaccination with ZH9 (21, 22, 31). All serum samples used in the opsonophagocytosis assays were heat treated (56°C; 30 min) to inactivate the complement system and remove its contribution to phagocytic uptake. Complement, therefore, should not play a role in uptake of S. Typhi in our assay, even if complement components are upregulated following vaccination. To confirm that serum immunoglobulins are the major contributors to the observed effects, we purified immunoglobulins from the sera tested in Fig. 1A using T-Gel, which binds immunoglobulins IgG, IgA, and IgM. We then measured the phagocytic uptake of S. Typhi opsonized with these purified immunoglobulins; approximately 11 ␮g of purified postvaccination immunoglobulins resulted in the greatest increases in phagocytic uptake of opsonized bacteria relative to day 0 and the placebo (Fig. 3). Changes in phagocytosis were negligible for immunoglobulins isolated from a placebo control at any time point; day 0 was similar to day 7, day 14, and day 28 (Fig. 3). Conversely, substantially more bacteria were phagocytosed

3191

at all time points (day 7, day 14, and day 28) than in autologous prevaccination samples when purified postvaccination immunoglobulins were added (Fig. 3). There was no significant difference in uptake between bacteria opsonized with purified immunoglobulins and bacteria opsonized with serum (P ⫽ 0.19). Therefore, immunoglobulins (IgG, IgA, and perhaps IgM) are the primary opsonins contributing to the increases in phagocytic uptake of S. Typhi in our assay. Uptake observed for samples opsonized with immunoglobulins from the day 7 samples appeared to be slightly lower than that of 5% serum suggesting there may be other opsonins besides antibodies contributing to uptake of the bacteria present in the serum. Of note, the total number of bacteria phagocytosed when opsonized with purified immunoglobulins was less than those opsonized with serum (approximately 2- to 4-fold [data not shown]). This was observed for the placebo, as well as for the vaccination samples, indicating, not surprisingly, that there are other components in the serum generally affecting phagocytosis of the bacteria but not contributing to the specific increases in uptake observed in the postvaccination sera. Therefore, S. Typhi-specific antibodies produced following ZH9 vaccination are largely responsible for increases in uptake of wild-type S. Typhi by human macrophages. Survival of wild-type S. Typhi opsonized with postvaccination sera is reduced in macrophages. Survival within phagocytic cells is an essential step in the development of systemic typhoid fever. Attenuation of S. Typhi strains can be measured by their ability to propagate within human macrophages (10, 11). Previous work has shown that the attenuated vaccine strain ZH9 is unable to replicate within the U-937 monocytic cell line when opsonized with human serum (unvaccinated but containing complement components) (38). Opsonins have also been shown to negatively affect Salmonella survival in mouse and human macrophages in vitro (12, 18). These results suggest that postvaccination opsonins could affect survival of wild-type S. Typhi within human macrophages. Bacteria are effectively phagocytosed when opsonized with postvaccination serum, as shown in Fig. 1 to 3; however, Salmonella species are often able to survive within the host’s macrophages. If the increases in uptake of bacteria are not concomitant with increases in killing, they could be detrimental to the host, potentially exacerbating the infection. Using a gentamicin survival assay, we examined whether opsonins could decrease survival and/or replication of S. Typhi within macrophages in addition to affecting phagocytic uptake. The survival of S. Typhi is defined as the number of bacteria present within macrophages after 48 h compared to identically treated bacteria within macrophages after 2 h. The survival of S. Typhi opsonized with immune serum is then divided by the survival of S. Typhi opsonized with preimmune serum to determine whether immune serum negatively affects survival and/or replication of the bacteria within macrophages (Fig. 4). Bacteria opsonized with prevaccination sera or no sera replicated after 48 h, demonstrating that S. Typhi is able to survive within human macrophages after opsonization with human serum if (i) complement is inactivated and (ii) prior exposure to S. Typhi has not occurred (day 0 serum). However, bacteria opsonized with postvaccination sera either died more rapidly or had severe defects in replication compared to day 0- or placebo-opsonized samples (P ⬍ 0.05). In particular, there was

3192

LINDOW ET AL.

FIG. 4. Wild-type S. Typhi has reduced survival in human macrophages when opsonized with postvaccination serum. To test for survival of S. Typhi within macrophages, S. Typhi opsonized with 5% heat-inactivated serum from prevaccination (day 0) or postvaccination (day 7, day 14, and day 28) or placebo (day 0, day 7, day 14, and day 28) time points were incubated with THP-1 macrophages at an MOI of 20 for 2 h and 48 h. Survival is equal to the number of bacteria at 48 h divided by the number at 2 h. The result is divided by the survival of the autologous day 0 sample. All samples were run in duplicate. n ⫽ 9 vaccinees or 7 placebos. *, the survival of the vaccine group differed from that of the placebo group (P ⬍ 0.05). Group-by-day differences approached significance (P ⫽ 0.06) for the vaccinee group compared to the placebo group. **, survival of immune day 7-opsonized bacteria was less than that of day 0-opsonized bacteria (P ⬍ 0.005) or placebo day 7-opsonized bacteria (P ⬍ 0.005).

a statistically significant difference in survival between day 7-opsonized S. Typhi and both day 0 (P ⬍ 0.01)- and placebo (P ⬍ 0.01)-opsonized bacteria, although the group-by-day interaction did not reach statistical significance (P ⫽ 0.06). No difference was observed for placebos at any time point relative to autologous day 0 samples. These data show that opsonins and/or the mechanism of uptake of S. Typhi into human macrophages affects survival and/or replication of the bacteria within macrophages. Within a week of vaccination, opsonins are present that are able to enhance macrophage-mediated killing of wild-type S. Typhi, in addition to increasing the number of bacteria phagocytosed by host macrophages. Antibodies have bactericidal activity. Recent work in Nepal demonstrated that individuals living in a region where typhoid is endemic have serum antibodies with bactericidal activity against S. Typhi (36). To further explore the functional activity of S. Typhi antibodies, we tested our samples for antibodymediated, complement-dependent killing using serum bactericidal assays (SBA), as previously described (36). The bactericidal antibody titer is defined as the inverse of the serum dilution at which ⱕ50% of the initial bacteria survive. Mean bactericidal antibody titers are significantly higher in the vaccination group than in placebo controls (P ⬍ 0.01) (Fig. 5). SBA assays using postvaccination sera demonstrated that mean bactericidal antibody titers are significantly higher than before vaccination (P ⬍ 0.05) (Fig. 5). SBA titers are also significantly higher at day 14 than at all other vaccination time points (P ⬍ 0.01). Interestingly, only 6 of 11 subjects’ sera examined had detectable SBA titers (titers ⱖ 250), although all of the subjects tested produced IgG and IgA antibodies specific to S. Typhi

INFECT. IMMUN.

FIG. 5. Bactericidal antibody titers are higher in postvaccination sera than in prevaccination or placebo samples. S. Typhi (250 CFU) was incubated with heat-inactivated, serially diluted serum (day 0, day 7, day 14, or day 28) and 5% baby rabbit complement. The bactericidal antibody titer equals the inverse of the serum dilution at which ⱕ50% of the bacteria survive relative to the no-serum control. Mean titers for vaccine day 0, day 7, day 14, and day 28 are 568 ⫾ 252, 2,023 ⫾ 848, 3,477 ⫾ 1,372, and 1,682 ⫾ 655, respectively. The mean titers for placebo samples are 50 ⫾ 50, 75 ⫾ 53, 125 ⫾ 100, and 75 ⫾ 53 for day 0, day 7, day 14, and day 28, respectively. All samples were run in duplicate. n ⫽ 11 vaccinees or 10 placebos. The vaccine titers differed from placebo titers (P ⬍ 0.01). *, in vaccinees, the day 14 titers were significantly different from those at all other time points (P ⬍ 0.01). Horizontal lines indicate mean titers, and error bars indicate SE.

(31). Two of the five sera that did not respond in this assay were analyzed for uptake and macrophage killing. Both resulted in increased uptake of S. Typhi at all postvaccination time points and reduced survival within macrophages when opsonized with day 7 and day 14 sera. Together, these data suggest that there are two antibody-dependent mechanisms for killing S. Typhi and that the antibodies required for each function may be distinct. DISCUSSION This work defines the functional role of S. Typhi-specific antibodies generated following vaccination. Identifying the clinically relevant functions of antibodies mounted against bacterial pathogens has been particularly challenging, because these antibodies generally opsonize bacteria rather than neutralizing them, as is the case for many viruses and toxins. Neutralizing antibodies often serve as the gold standard for correlates of protection for viruses and bacterial toxins, which offers a clear marker for measuring immune response, a marker that is lacking in S. Typhi infections. Moreover, no dominant epitopes have been identified for S. Typhi, which makes the search for any correlate of protection even more difficult. Studies of natural typhoid infection and vaccination have measured serum levels of S. Typhi antibodies (e.g., antiVi, anti-LPS, or anti-flagellin) as markers of immunogenicity in lieu of a correlate of protection. Yet, despite extensive data analysis of antibody titers following typhoid infection or vaccination, it has been difficult to establish the role that antibodies play in clearance of typhoid fever (7, 8, 13, 14, 20, 23–26, 28, 32, 34, 39, 46). A recent Nepalese study failed to show a correlation between bactericidal antibody activity and anti-Vi antibody titers, suggesting that some of the antibodies that have been the focus of typhoid studies for decades may or may not be functionally relevant to clearance of S. Typhi infections

VOL. 79, 2011

POSTVACCINATION ANTIBODIES KILL SALMONELLA TYPHI

(36). The assays described in our work, if implemented in vaccine or natural typhoid studies, could aid in vaccine development by analyzing whether S. Typhi-specific antibodies have bactericidal roles, rather than measuring antibody titers. The resulting data could advance the field toward defining a relative correlate of protection for S. Typhi. This work contributes three major findings to the field by describing mechanisms the immune system can utilize to kill S. Typhi: (i) opsonins generated following vaccination with ZH9 increase the uptake of wild-type S. Typhi by human macrophages and enhance the killing of phagocytosed bacteria, (ii) the primary opsonins responsible for these activities are antibodies, and (iii) vaccination also generates S. Typhi-specific antibodies that are recognized by complement components and that aid in killing S. Typhi. First, utilizing samples from a vaccine trial of ZH9 (a nextgeneration oral typhoid vaccine candidate from Emergent Biosolutions), we demonstrated that opsonization with postvaccination sera augments both uptake and killing of S. Typhi by macrophages. Gentamicin survival assays and phagocytic-index measurements clearly linked opsonins, predominately immunoglobulins, generated as early as 7 days postvaccination to the following functions: significantly increased phagocytosis of wild-type S. Typhi; significantly enhanced killing of the bacteria by macrophages; and, potentially, diminished replication of the bacteria within macrophages. Peak uptake and macrophage-killing activities were noted for day 7-opsonized S. Typhi. We also showed the importance of antibody-mediated, complement-dependent killing of S. Typhi using serum bactericidal assays. S. Typhi-specific antibodies generated postvaccination are recognized by complement in order to facilitate lysis of the bacteria by complement components. The peak of bactericidal antibody activity occurred 2 weeks postvaccination, correlating with peak IgG levels and suggesting that S. Typhi-specific IgGs are largely responsible for complementmediated killing (31). Similar studies measuring vibriocidal antibody responses following exposure to V. cholerae implicated anti-LPS IgMs as the primary antibody subtype contributing to complement-mediated cell lysis (5, 16, 29, 48). The IgM response to S. Typhi has heretofore been uncharacterized. Future work should address the subtype of antibody responsible for complement-mediated killing of S. Typhi. Our data are supported by studies in mice in which opsonization of Salmonella spp. with anti-Salmonella antibodies led to increased bacterial killing (3, 12, 18, 45, 51). Recent work by Wilson et al. also supports our findings that specific antibody responses can aid in clearance of systemic S. Typhi infections and may be of particular import, as the Vi capsule allows S. Typhi to evade complement detection in the blood of mice (51). Furthermore, their work demonstrates a clear role for complement in the binding of S. Typhi to complement receptors on mouse macrophages, indicating that complement is likely to be involved in clearance of S. Typhi (51). Although this study represents a significant step forward in delineating the functional roles of antibodies in humans, further human population-based work is necessary to establish the relative importance of antibody isotypes for each function, to determine the kinetics of opsonization responses, and to explore the contributions of human complement components to each function described. Given limited serum volumes, we

3193

were unable to identify which antibody isotype is predominately responsible for opsonization in this system. The kinetics of the response strongly suggest that IgM, and possibly IgA, is responsible for key activities at early time points (day 7), whereas IgG predominates by day 14 postvaccination. The geometric mean titers of S. Typhi-specific antibodies demonstrated that the antibodies peaked at day 14 for IgG and were high at day 7 and day 14 for IgA (31). Increases in uptake were observed with day 28 sera, suggesting that IgG- and perhaps IgA-mediated effects are maintained for at least a month following vaccination. Interestingly, non-typhoid-specific increases in phagocytosis were also observed (day 0-opsonized versus unopsonized bacteria). These data suggest serum proteins, such as mannose binding lectin, which would nonspecifically recognize S. Typhi, may be responsible for nonspecific uptake (19). The bactericidal activity of anti-Vi (capsule) antibodies is predominantly IgG2 mediated (36). Early work on the typhoid vaccine Ty21a showed that IgA played a role in antibodydependent cell cytotoxicity of S. Typhi (9, 42, 43). As only IgG antibodies have been analyzed in opsonization studies thus far, the role of IgA in opsonization and complement-mediated killing should be further explored. This is particularly important given that both IgG and IgA S. Typhi-specific LPS antibodies were detected in a majority of ZH9 vaccinees, suggesting that both classes of antibodies may play a role in protection against S. Typhi infections, particularly after oral vaccination (21, 31). It is also possible that different functions, such as opsonization and antibody-mediated, complement-dependent killing of bacteria, may be attributable to different classes of antibodies. Future studies will define the specific roles of IgG and IgA in the killing of S. Typhi and characterize the mechanisms of protection for the minority of subjects who lack a bactericidal antibody response. Similarly, for the identification of a relative correlate of protection, further work to better define the kinetics and relevant significance of opsonization and complement-mediated killing within the context of the overall human immune response to S. Typhi infection is still required. Cell-mediated immune responses, in addition to antibody responses and innate immunity, are clearly involved in the response to S. Typhi infection, but it is still unclear if all are required to clear the infection, if there is a temporal requirement for each, and whether the type of response is dependent on the individual (27, 40, 44, 47, 50). Additionally, we have noted the significance of nonantibody, serum-opsonizing factors in the serum, which also need identification and characterization. Using a novel collection of specimens from human vaccinees, this work has identified functional roles for antibodies and expanded our understanding of antibody-dependent complement killing in S. Typhi vaccination. In conjunction with recent advances in the understanding of the cellular immune responses, these data advance our understanding of which immunologic components contribute to resolution of infection and assist in establishment of protection following natural typhoid infection or vaccination. ACKNOWLEDGMENTS We thank all the members of the UVM Vaccine Testing Center clinical and laboratory teams. We thank Christopher Huston for the

3194

LINDOW ET AL.

generous use of his microscope (R01 AI072021-02S1). We are grateful to Emergent Biosolutions for use of M01ZH09 serum samples. We owe a great deal of thanks to Sergio Grinstein and Manuella Raffatellu for their technical help. We are grateful to M. Berkman, D. Monack, and R. Valdivia for gifts of plasmids. This work was supported by NCRR COBRE grant P20 RR 0121905. REFERENCES 1. Bhutta, Z. A. 1996. Impact of age and drug resistance on mortality in typhoid fever. Arch. Dis. Child. 75:214–217. 2. Bhutta, Z. A., and J. Threlfall. 2009. Addressing the global disease burden of typhoid fever. JAMA 302:898–899. 3. Biozzi, G., C. Stiffel, B. N. Halpern, L. Le Minor, and D. Mouton. 1961. Measurement of the opsonic effect of normal and immune sera on the phagocytosis of Salmonella Typhi by the reticuloendothelial system. J. Immunol. 87:296–300. 4. Canh, D. G., et al. 2004. Effect of dosage on immunogenicity of a Vi conjugate vaccine injected twice into 2- to 5-year-old Vietnamese children. Infect. Immun. 72:6586–6588. 5. Clemens, J. D., et al. 1991. Field trial of oral cholera vaccines in Bangladesh: serum vibriocidal and antitoxic antibodies as markers of the risk of cholera. J. Infect. Dis. 163:1235–1242. 6. Crump, J. 2004. The global burden of typhoid fever. Bull. World Health Organ. 82:346–353. 7. Cryz, S. J., Jr., E. Furer, and M. M. Levine. 1988. Effectiveness of oral, attenuated live Salmonella typhi Ty 21a vaccine in controlled field trials. Schweiz. Med. Wochenschr. 118:467–470. 8. Cryz, S. J., Jr., et al. 1993. Safety and immunogenicity of Salmonella typhi Ty21a vaccine in young Thai children. Infect. Immun. 61:1149–1151. 9. D’Amelio, R., et al. 1988. Comparative analysis of immunological responses to oral (Ty21a) and parenteral (TAB) typhoid vaccines. Infect. Immun. 56:2731–2735. 10. Dragunsky, E. M., E. Rivera, H. D. Hochstein, and I. S. Levenbook. 1990. In vitro characterization of Salmonella typhi mutant strains for live oral vaccines. Vaccine 8:263–268. 11. Dragunsky, E. M., C. R. Wooden, S. A. Vargo, and I. S. Levenbook. 1989. Salmonella typhi vaccine strain in vitro; low infectivity in human cell line U937. J. Biol. Stand. 17:353–360. 12. Drecktrah, D., L. A. Knodler, R. Ireland, and O. Steele-Mortimer. 2006. The mechanism of Salmonella entry determines the vacuolar environment and intracellular gene expression. Traffic 7:39–51. 13. Engels, E. A., M. E. Falagas, J. Lau, and M. L. Bennish. 1998. Typhoid fever vaccines: a meta-analysis of studies on efficacy and toxicity. BMJ 316:110– 116. 14. Fraser, A., M. Paul, E. Goldberg, C. J. Acosta, and L. Leibovici. 2007. Typhoid fever vaccines: systematic review and meta-analysis of randomised controlled trials. Vaccine 25:7848–7857. 15. Galen, J. E., et al. 2009. Salmonella enterica serovar Typhi live vector vaccines finally come of age. Immunol. Cell Biol. 87:400–412. 16. Glass, R. I., et al. 1985. Seroepidemiological studies of El Tor cholera in Bangladesh: association of serum antibody levels with protection. J. Infect. Dis. 151:236–242. 17. Hohmann, E. L., C. A. Oletta, K. P. Killeen, and S. I. Miller. 1996. phoP/ phoQ-deleted Salmonella typhi (Ty800) is a safe and immunogenic singledose typhoid fever vaccine in volunteers. J. Infect. Dis. 173:1408–1414. 18. Ishibashi, Y., and T. Arai. 1996. A possible mechanism for host-specific pathogenesis of Salmonella serovars. Microb. Pathog. 21:435–446. 19. Jack, D. L., et al. 1998. Activation of complement by mannose-binding lectin on isogenic mutants of Neisseria meningitidis serogroup B. J. Immunol. 160: 1346–1353. 20. Keitel, W. A., N. L. Bond, J. M. Zahradnik, T. A. Cramton, and J. B. Robbins. 1994. Clinical and serological responses following primary and booster immunization with Salmonella typhi Vi capsular polysaccharide vaccines. Vaccine 12:195–199. 21. Kirkpatrick, B. D., et al. 2006. Evaluation of Salmonella enterica serovar Typhi (Ty2 aroC⫺ssaV⫺) M01ZH09, with a defined mutation in the Salmonella pathogenicity island 2, as a live, oral typhoid vaccine in human volunteers. Vaccine 24:116–123. 22. Kirkpatrick, B. D., et al. 2005. The novel oral typhoid vaccine M01ZH09 is well tolerated and highly immunogenic in 2 vaccine presentations. J. Infect. Dis. 192:360–366. 23. Klugman, K. P., H. J. Koornhof, J. B. Robbins, and N. N. Le Cam. 1996. Immunogenicity, efficacy and serological correlate of protection of Salmonella typhi Vi capsular polysaccharide vaccine three years after immunization. Vaccine 14:435–438. 24. Levine, M. M., et al. 1999. Duration of efficacy of Ty21a, attenuated Salmonella typhi live oral vaccine. Vaccine 17(Suppl. 2):S22–S27.

Editor: A. J. Ba¨umler

INFECT. IMMUN. 25. Levine, M. M., C. Ferreccio, S. Cryz, and E. Ortiz. 1990. Comparison of enteric-coated capsules and liquid formulation of Ty21a typhoid vaccine in randomised controlled field trial. Lancet 336:891–894. 26. Levine, M. M., D. Hone, C. Tacket, C. Ferreccio, and S. Cryz. 1990. Clinical and field trials with attenuated Salmonella typhi as live oral vaccines and as “carrier” vaccines. Res. Microbiol. 141:807–816. 27. Levine, M. M., C. O. Tacket, and M. B. Sztein. 2001. Host-Salmonella interaction: human trials. Microbes Infect. 3:1271–1279. 28. Lin, F. Y., et al. 2001. The efficacy of a Salmonella typhi Vi conjugate vaccine in two-to-five-year-old children. N. Engl. J. Med. 344:1263–1269. 29. Losonsky, G. A., et al. 1996. Factors influencing secondary vibriocidal immune responses: relevance for understanding immunity to cholera. Infect. Immun. 64:10–15. 30. Lundgren, A., J. Kaim, and M. Jertborn. 2009. Parallel analysis of mucosally derived B- and T-cell responses to an oral typhoid vaccine using simplified methods. Vaccine 27:4529–4536. 31. Lyon, C. E., et al. 2010. In a randomized, double-blinded, placebo-controlled trial, the single oral dose typhoid vaccine, M01ZH09, is safe and immunogenic at doses up to 1.7 x 10(10) colony-forming units. Vaccine 28:3602– 3608. 32. Mai, N. L., et al. 2003. Persistent efficacy of Vi conjugate vaccine against typhoid fever in young children. N. Engl. J. Med. 349:1390–1391. 33. Mousa, G. Y., J. R. Trevithick, J. Bechberger, and D. G. Blair. 1978. Cytochalasin D induces the capping of both leukaemia viral proteins and actin in infected cells. Nature 274:808–809. 34. Olanratmanee, T., M. Levine, G. Losonsky, V. Thisyakorn, and S. J. Cryz, Jr. 1992. Safety and immunogenicity of Salmonella typhi Ty21a liquid formulation vaccine in 4- to 6-year-old Thai children. J. Infect. Dis. 166:451–452. 35. Pegues, D. A., M. E. Ohl, and S. I. Miller. 2004. Salmonella species, including Salmonella Typhi, p. 2636–2653. In G. L. Mandell, J. E. Bennett, and R. Dolin (ed.), Principles and practice of infectious disease. Churchill-Livingstone, Philadelphia PA. 36. Pulickal, A. S., et al. 2009. Kinetics of the natural, humoral immune response to Salmonella enterica Serovar Typhi in Kathmandu, Nepal. Clin. Vaccine Immunol. 16:1413–1419. 37. Rowe, B., L. R. Ward, and E. J. Threlfall. 1997. Multidrug-resistant Salmonella typhi: a worldwide epidemic. Clin. Infect. Dis. 24(Suppl. 1):S106–S109. 38. Stratford, R., et al. 2005. Optimization of Salmonella enterica serovar typhi DeltaaroC DeltassaV derivatives as vehicles for delivering heterologous antigens by chromosomal integration and in vivo inducible promoters. Infect. Immun. 73:362–368. 39. Sur, D., et al. 2009. A cluster-randomized effectiveness trial of Vi typhoid vaccine in India. N. Engl. J. Med. 361:335–344. 40. Sztein, M. B. 2007. Cell-mediated immunity and antibody responses elicited by attenuated Salmonella enterica Serovar Typhi strains used as live oral vaccines in humans. Clin. Infect. Dis. 45(Suppl. 1):S15–S19. 41. Tacket, C. O., and M. M. Levine. 2007. CVD 908, CVD 908-htrA, and CVD 909 live oral typhoid vaccines: a logical progression. Clin. Infect. Dis. 45(Suppl. 1):S20–S23. 42. Tagliabue, A., et al. 1985. Cellular immunity against Salmonella typhi after live oral vaccine. Clin. Exp. Immunol. 62:242–247. 43. Tagliabue, A., et al. 1986. IgA-driven T cell-mediated anti-bacterial immunity in man after live oral Ty 21a vaccine. J. Immunol. 137:1504–1510. 44. Tam, M. A., A. Rydstrom, M. Sundquist, and M. J. Wick. 2008. Early cellular responses to Salmonella infection: dendritic cells, monocytes, and more. Immunol. Rev. 225:140–162. 45. Tobar, J. A., P. A. Gonzalez, and A. M. Kalergis. 2004. Salmonella escape from antigen presentation can be overcome by targeting bacteria to Fc gamma receptors on dendritic cells. J. Immunol. 173:4058–4065. 46. Tran, T. H., et al. 2010. A randomised trial evaluating the safety and immunogenicity of the novel single oral dose typhoid vaccine M01ZH09 in healthy Vietnamese children. PLoS One 5:e11778. 47. Wahid, R., et al. 2011. Oral priming with Salmonella Typhi vaccine strain CVD 909 followed by parenteral boost with the S. Typhi Vi capsular polysaccharide vaccine induces CD27(⫹)IgD(-) S. Typhi-specific IgA and IgG B memory cells in humans. Clin. Immunol. 138:187–200. 48. Wasserman, S. S., et al. 1994. Kinetics of the vibriocidal antibody response to live oral cholera vaccines. Vaccine 12:1000–1003. 49. WHO. 2003. posting date. Background document: the diagnosis, treatment and prevention of typhoid fever. WHO WHO/V&B/03.07. http://www.who .int/vaccines-documents. 50. Wick, M. J. 2007. Monocyte and dendritic cell recruitment and activation during oral Salmonella infection. Immunol. Lett. 112:68–74. 51. Wilson, R. P., et al. 2011. The Vi capsular polysaccharide prevents complement receptor 3-mediated clearance of Salmonella enterica serotype Typhi. Infect. Immun. 79:830–837.