This work was supported by 1R01AI125560-01 and start-up funds from The Ohio State University.

The authors have nothing to disclose.

The comprehensive protocol described here to study vaccine-induced immunity to B. pertussis infection will also permit evaluation of host responses to a variety of other pathogens. The protocol discusses methods to deliver immunizations, determine vaccine efficacy following pathogen challenge, and parallel dissection of immune function. In adapting the protocol in order to study other pathogens, several parameters would need to be modified. These include, but are not limited to, the mode of animal anesthesia, vaccine composition, dose, and route of administration. In addition, the dose and route of administration of the challenged pathogen of interest, selected tissue for harvest, and downstream evaluation of immune responses are factors that may be modified for the pathogen/disease of interest.
The mouse model of B. pertussis infection is widely used and is an excellent model for pathogenesis and vaccinology studies16,17,18,19,20. Murine models exhibit many similarities to human infection, including: i) bacteria are limited to the respiratory tract and multiply rapidly, ii) young animals display comparatively severe infections, iii) good correspondence between vaccines that protect children against pertussis and those that protect mice against infection, and iv) B. pertussis-specific T-cells and antibodies mediate natural and vaccine-induced protective immunity21,22,23,24. Therefore, the murine model permits the study of immunization effects on bacterial clearance25. Another advantage of using mice to model B. pertussis infections is the availability of genetically modified knockout and transgenic animals to study critical players in vaccine-induced immune responses19,26.
The majority of immunizations are administered parenterally, specifically via the intramuscular (i.m.) route. This route is preferred because it elicits systemic immunity with minimal local reactogenicity27,28,29. Alternatives to intramuscular injections include subcutaneous (s.c.) or intraperitoneal (i.p.) delivery, which also induce systemic responses. Subcutaneous routes are also attractive, as there is a large population of resident antigen presenting cells (APC) within the dermis with access to the vascular and lymphatic systems30. However, intradermal delivery has not been explored for the pertussis vaccine. Intraperitoneal delivery of experimental aPV generates similar immunity to intramuscular immunization31 and is a reliable injection route for murine studies, albeit impractical for clinical use.
This protocol utilizes the i.m. immunization route, which provides protection in the lungs, but not the nasopharynx32. Recent studies have shown that an intranasal (i.n.) vaccine promotes a local, mucosal immune response that protects the upper and lower respiratory tracts, particularly the nose, which serves as a reservoir of bacteria that may be subsequently transmitted to other hosts33. In addition, i.n. immunization has been shown to generate tissue resident memory that is not generated by systemic immunization33,34. Therefore, the choice of vaccine administration route depends greatly on the type of vaccine and immune response required to protect against disease.
The intranasal route of bacterial delivery described in this protocol is a widely used method for respiratory pathogens that safely and consistently delivers a bacterial inoculum directly into the respiratory tract of anesthetized mice. Our protocol uses a high-volume dose (40-50 &#181;L) that is inhaled into the nasopharynx and travels into the lower respiratory tract. A low volume inoculum (10-20 &#181;L) would colonize the nasopharynx, but colonization of the lungs will be minimal35. However, inhalation of B. pertussis-containing air droplets is considered the natural mode of infection of individuals and transmission. This route of administration has been used in various animal models36,37. To achieve comparable levels of infection to direct i.n. inoculation, considerably higher concentrations of the bacterial inoculum (109-1011 CFUs/mL) and delivery time are required38.
To establish colonization of mouse respiratory tract, this protocol uses fresh bacteria, grown in liquid culture to ensure that bacteria used for inoculation are in the virulent phase and are replicating in the log stage. Experimenters can also use an inoculum from pre-titrated, frozen stocks. This enables an knowledge of the CFUs being administered to the mouse. However, the bacteria in these inoculums may be in the non-virulent phase, which can reduce the efficiency of respiratory tract colonization39. After the infection, the inoculum is plated in order to determine the bacterial CFUs delivered to the mouse. Investigators may also choose to plate the inoculum prior to the in vivo infection to ensure viability of the bacteria and confirm dose of the inoculum.
After infection, bacterial CFUs are enumerated by homogenizing isolated tissue from sacrificed mice at a predetermined timepoint. A disadvantage to this method is its inability to kinetically track pathogen CFUs for a specified set of mice. Researchers must use multiple groups of animals sacrificed at different timepoints to examine vaccine-induced bacterial restriction. The number of mice in each experiment will vary depending on the number of timepoints to examine and the desired effect size. This may introduce increased biological variation. Integration of a bioluminescent or fluorescent reporter gene into the pathogen of interest will permit non-invasive monitoring of growth and dissemination in vivo throughout the course of infection35. This method reduces biological variation and minimizes the required number of experimental animals, since longitudinal data are obtained from the same group of animals.
The tissue dissociation and homogenization protocol employed here for respiratory tract tissues is also applicable for colony enumeration of other solid tissues such as liver, kidney, intestine, and/or bladder to interrogate sites of infection and dissemination of pathogens of interest.
Along with CFU enumeration, this protocol enables characterization of immune responses elicited by immunization and natural infection. Specifically, quantification of cytokines produced systemically and locally allow for the determination of efficacious immune profiles resulting from immunization. Cytokines are immunomodulators that promote cellular influx to affected tissues and activation of cellular immunity, as well as provide help for generation of humoral responses10,40,41. Using this protocol, cytokines produced in various tissue compartments, such as the lung and spleen, are detected. Although not shown here, the stimulation protocol is also applicable for evaluation of responses in the draining lymph nodes.
ELISA analysis allows detection and quantification of cytokines in media supernatants or mixed suspensions (i.e., homogenates or serum), yielding information at the population level and characterizing antigen-specific cytokine responses from a mixed cell culture at designated timepoints. In contrast, methods like ELISPOT or flow cytometry permit evaluation of the number of cells that produce an analyte and the level of expression per cell42,43. Although not described here, these methods are also applicable to detection of antigen-specific B cells. The combination of CFU enumeration and immunological assays provide complete picture of the response to immunization.
Other analyses that may be performed using this infection protocol include epigenetic or transcriptomic analysis when DNA and RNA is obtained from tissues of interest44. Thus, with consideration of the appropriate vaccine composition, immunization, and pathogen delivery route, this protocol is versatile in its implementation and easily suited for various models of infection. These techniques are also applicable to non-infectious diseases (i.e., cancer, multiple sclerosis, asthma, autoimmune diseases) where vaccines are used as a therapeutic modality.

Vaccines represent one of the greatest public health achievements of the 20th century, yet we still do not fully understand the mechanisms by which successful vaccines stimulate protective immunity. The identification of molecular signatures (e.g., cell activation markers, expansion of cellular subtypes, and patterns of gene expression) induced after vaccination provides a plethora of information for predicting and generating an efficacious immune response. The complexity of host-pathogen responses cannot be adequately replicated using in vitro cell culture systems1. In vivo vaccine models are designed to concomitantly evaluate multiple immune cell types within the host. This provides an advantage when characterizing vaccine antigen processing and presentation, differential cytokine secretion, and expansion of immune cells. The protocol described here provides a detailed method to determine vaccine efficacy through evaluation of the systemic and local immune responses and quantification of pathogen burden in tissues of interest. The example provided here tests the efficacy of an experimental vaccine for the pathogen Bordetella pertussis (B. pertussis).
B. pertussis is a gram-negative bacterium that is the etiological agent of the respiratory disease whooping cough (pertussis)2,3. Close contact with infected individuals (symptomatic or asymptomatic) leads to transmission, colonization, and disease. Despite significant global vaccine coverage4, pertussis is considered a resurging disease in many nations around the world and is a major cause of preventable childhood deaths5,6,7,8. In 2015, B. pertussis and pertussis were included in the National Institute of Allergy and infectious Diseases (NIAID) emerging infectious pathogen/disease list, emphasizing the need for development of a better vaccine that confers long-lived protective immunity.
Currently, an active area of investigation to control pertussis resurgence is development of a next-generation acellular pertussis vaccine (aPV) with an optimal combination of novel adjuvants and antigens to mimic the immune response elicited by the whole-cell pertussis vaccine (wPV)9. Using the protocol described, we recently reported that the modification of a current FDA-approved aPV by the addition of a novel adjuvant, Bordetella colonization factor A (BcfA), resulted in more efficient reduction of B. pertussis bacterial load from mouse lungs10,11. This increased protection was accompanied by the skewing of an alum-induced Th1/Th2 immune response to the more protective Th1/Th17 immune profile10. This protocol is detailed and comprehensive, enabling the investigator to obtain maximal information through concurrent evaluation of host and immune responses to a variety of pathogens.
The protocol described here follows the representative vaccine schedule, shown in Figure 1, to ensure optimal host immune responses.

<table border="0" cellpadding="0" cellspacing="0" style="width:933px;" width="933">
	<colgroup>
		<col />
		<col />
		<col />
		<col />
	</colgroup>
	<tbody>
		<tr height="21">
			<td height="21" style="height:21px;width:333px;">2L induction chamber</td>
			<td style="width:175px;">Vet Equip</td>
			<td style="width:119px;">941444</td>
			<td style="width:307px;"></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:333px;">Fluriso</td>
			<td style="width:175px;">Vet One</td>
			<td style="width:119px;">V1 501017</td>
			<td>any brand is appropriate</td>
		</tr>
		<tr height="25">
			<td height="25" style="height:25px;">Bordet Gengou Agar Base</td>
			<td style="width:175px;">BD bioscience</td>
			<td style="width:119px;">248200</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:333px;">Casein</td>
			<td style="width:175px;">Sigma</td>
			<td style="width:119px;">C-7078</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:333px;">Casamino acids</td>
			<td style="width:175px;">VWR</td>
			<td style="width:119px;">J851-500G</td>
			<td>Strainer Scholte (SS) media components</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:333px;">L-Glutamic acid</td>
			<td style="width:175px;">Research Products Int</td>
			<td style="width:119px;">G36020-500</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:333px;">L-Proline</td>
			<td style="width:175px;">Research Products Int</td>
			<td style="width:119px;">P50200-500</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:333px;">Sodium Chloride</td>
			<td style="width:175px;">Fisher</td>
			<td style="width:119px;">BP358-10</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:333px;">Potassium Phosphate monobasic</td>
			<td style="width:175px;">Fisher</td>
			<td style="width:119px;">BP362-1</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:333px;">Potassium Chloride</td>
			<td style="width:175px;">Fisher</td>
			<td style="width:119px;">P217-500</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:333px;">Magnesium Chloride hexahydrate</td>
			<td style="width:175px;">Fisher</td>
			<td style="width:119px;">M2670-500G</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:333px;">Calcium Chloride</td>
			<td style="width:175px;">Fisher</td>
			<td style="width:119px;">C75-500</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:333px;">Tris base</td>
			<td style="width:175px;">Fisher</td>
			<td style="width:119px;">BP153-1</td>
			<td></td>
		</tr>
		<tr height="23">
			<td height="23" style="height:23px;width:333px;">L-cysteine HCl</td>
			<td style="width:175px;">Fisher</td>
			<td style="width:119px;">BP376-100</td>
			<td>SS media suplements</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:333px;">Ferrous Sulfate heptahydrate</td>
			<td style="width:175px;">Sigma</td>
			<td style="width:119px;">F-7002</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:333px;">Niacin</td>
			<td style="width:175px;">Research Products Int</td>
			<td style="width:119px;">N20080-100</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:333px;">Glutathione</td>
			<td style="width:175px;">Research Products Int</td>
			<td style="width:119px;">G22010-25</td>
			<td></td>
		</tr>
		<tr height="26">
			<td height="26" style="height:26px;width:333px;">Ascorbic acid</td>
			<td style="width:175px;">Research Products Int</td>
			<td style="width:119px;">A50040-500</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:333px;">RPMI 1640</td>
			<td style="width:175px;">ThermoFisher Scientific</td>
			<td>11875093</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:333px;">FBS</td>
			<td style="width:175px;">Sigma</td>
			<td style="width:119px;">F2442-500mL</td>
			<td>&#160;any US source, non-heat inactivated</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:333px;">gentamicin</td>
			<td style="width:175px;">ThermoFisher Scientific</td>
			<td>15710064</td>
			<td style="width:307px;"></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:333px;">B-mercaptoethanol</td>
			<td style="width:175px;">Fisher&#160;</td>
			<td style="width:119px;">BP176-100</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:333px;">15mL dounce tissue grinder</td>
			<td style="width:175px;">Wheaton</td>
			<td>357544</td>
			<td>any similar brand is appropriate</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:333px;">Cordless Hand Homogenizer</td>
			<td style="width:175px;">Kontes/Sigma&#160;</td>
			<td style="width:119px;">Z359971-1EA</td>
			<td>any similar brand is appropriate</td>
		</tr>
		<tr height="42">
			<td height="42" style="height:42px;width:333px;">Instruments - scissors, curve scissors, forceps, fine forceps, triangle spreaders</td>
			<td style="width:175px;"></td>
			<td style="width:119px;"></td>
			<td>any brand is appropriate</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:333px;">3mL syringes</td>
			<td style="width:175px;">BD bioscience</td>
			<td style="width:119px;">309657</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:333px;">15mL conical tubes</td>
			<td style="width:175px;">Fisher&#160;</td>
			<td style="width:119px;">339651</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:333px;">1.5mL microfuge tubes</td>
			<td style="width:175px;">Denville</td>
			<td style="width:119px;">C2170</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:333px;">70um cell strainers</td>
			<td style="width:175px;">Fisher&#160;</td>
			<td style="width:119px;">22363548</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:333px;">60mm plates</td>
			<td style="width:175px;">ThermoFisher Scientific</td>
			<td style="width:119px;">130181</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:333px;">48-well tissue culture plates</td>
			<td style="width:175px;">ThermoFisher Scientific</td>
			<td>08-772-1C</td>
			<td></td>
		</tr>
		<tr height="42">
			<td height="42" style="height:42px;width:333px;">1mL insulin syringe 28G1/2</td>
			<td style="width:175px;">Fisher Scientific/Excel Int.</td>
			<td style="width:119px;">14-841-31</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:333px;">Mouse IFN-gamma ELISA Ready-SET-Go! Kit</td>
			<td style="width:175px;">Invitrogen / eBioscience</td>
			<td style="width:119px;">50-173-21</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:333px;">Mouse IL-17 ELISA Ready-SET-Go! Kit</td>
			<td style="width:175px;">Invitrogen / eBioscience</td>
			<td style="width:119px;">50-173-77</td>
			<td></td>
		</tr>
		<tr height="22">
			<td height="22" style="height:22px;width:333px;">Mouse IL-5 ELISA Ready-SET-Go! Kit</td>
			<td style="width:175px;">Invitrogen / eBioscience</td>
			<td style="width:119px;">50-172-09</td>
			<td></td>
		</tr>
	</tbody>
</table>

evaluation of host-pathogen responses and vaccine efficacy in mice

Vaccines are a 20th century medical marvel. They have dramatically reduced the morbidity and mortality caused by infectious diseases and contributed to a striking increase in life expectancy around the globe. Nonetheless, determining vaccine efficacy remains a challenge. Emerging evidence suggests that the current acellular vaccine (aPV) for Bordetella pertussis (B. pertussis) induces suboptimal immunity. Therefore, a major challenge is designing a next-generation vaccine that induces protective immunity without the adverse side effects of a whole-cell vaccine (wPV). Here we describe a protocol that we used to test the efficacy of a promising, novel adjuvant that skews immune responses to a protective Th1/Th17 phenotype and promotes a better clearance of a B. pertussis challenge from the murine respiratory tract. This article describes the protocol for mouse immunization, bacterial inoculation, tissue harvesting, and analysis of immune responses. Using this method, within our model, we have successfully elucidated crucial mechanisms elicited by a promising, next-generation acellular pertussis vaccine. This method can be applied to any infectious disease model in order to determine vaccine efficacy.

The model described shows a method to evaluate vaccine efficiency and immune responses during host-pathogen interactions. Figure 1 depicts the representative vaccine schedule used to immunize and infect mice and harvest tissues for analysis. Figure 2 demonstrates the setup of the anesthesia system employed to induce mice, enabling investigators to deliver immunizations and bacterial inoculums. Figure 3 shows example OD600 measurements and calculations to achieve a 1 OD bacterial suspension to prepare 100-fold diluted bacterial inoculum to be delivered to mice. Figure 4 depicts the inoculum preparation used for the infection, the plating scheme of the serial dilutions, and the example calculations used to enumerate CFUs delivered to mice based upon plating serial dilutions. Figure 5 shows antigen-specific recall responses elicited after seven days of stimulation with the vaccine antigen FHA. Immunize spleen cells from the combination vaccine group, Alum/FHA/BcfA, produced IFN&#947; and IL-17, while significantly downregulating IL-5. This effect promotes a Th1/Th17 polarization of the immune response during B. pertussis infection. Background levels of cytokines were produced by incubation of immunized spleen cells with media alone, indicating that the cytokines detected were recall responses to the immunization (data not shown). Figure 6 depicts an example CFU enumeration of bacteria recovered from respiratory tract tissues of an immunized mouse, using serial dilutions of B. pertussis tissue homogenates plated on 10% BG + Sm plates. Raw counts were multiplied by the dilution factor and total volume of the tissue lysate and the data were log transformed. CFUs from immunized animals are then compared to non-immunized (na&#239;ve) animals to determine the protective effect of the vaccine.
<img alt="Figure 1" class="xfigimg" src="/files/ftp_upload/58930/58930fig1.jpg" /> 
Figure 1: Representative vaccine schedule. Mice are immunized on day 0 and then boosted ~4 weeks later (d28) with the appropriate aPV. Infection of the mice with B. pertussis occurs ~2 weeks (d42) after boosting mice. After infection the animals are killed on various days (d43-56) post-inoculation. Tissue and blood is collected for downstream analysis (e.g., CFU enumeration, cytokine and antibody ELISAs). <a href="https://www-jove-com.remotexs.ntu.edu.sg/files/ftp_upload/58930/58930fig1large.jpg" target="_blank">Please click here to view a larger version of this figure.</a>
<img alt="Figure 2" class="xfigimg" src="/files/ftp_upload/58930/58930fig2.jpg" /> 
Figure 2:&#160;Anesthesia machine setup. Shown is an anesthetic vaporizer with attached induction chamber and scavenger system (inside biological safety cabinet). <a href="https://www-jove-com.remotexs.ntu.edu.sg/files/ftp_upload/58930/58930fig2large.jpg" target="_blank">Please click here to view a larger version of this figure.</a>
<img alt="Figure 3" class="xfigimg" src="/files/ftp_upload/58930/58930fig3.jpg" /> 
Figure 3: Preparation of 1 OD600 bacterial suspension. OD600 values were obtained from diluted primary B. pertussis cultures. One culture in log phase was used to calculate volume needed to obtain a culture at 1 OD600 culture for infection. Briefly, an OD600 of 1 was divided by the calculated OD600 of the desired culture to obtain a volume equaling 1 OD to be used for infection.&#160;Briefly, an OD600 of 1 was divided by the calculated OD600 of the desired culture to obtain a volume equaling 1 OD to be used for infection.&#160;<a href="https://www-jove-com.remotexs.ntu.edu.sg/files/ftp_upload/58930/58930fig3large.jpg" target="_blank">Please click here to view a larger version of this figure.</a>
<img alt="Figure 4" class="xfigimg" src="/files/ftp_upload/58930/58930fig4.jpg" /> 
Figure 4: Calculation of delivered intranasal inoculum. (A) Schematic of serial dilution of infection inoculum that is diluted to 10-4, 10-5, and 10-6. These dilutions are plated on 10% BG + Sm, incubated for 4 days at 37 &#176;C and then counted. (B) Counts from 10-4, 10-5, and 10-6 dilutions are then used to calculate intranasally delivered CFUs. <a href="https://www-jove-com.remotexs.ntu.edu.sg/files/ftp_upload/58930/58930fig4large.jpg" target="_blank">Please click here to view a larger version of this figure.</a>
<img alt="Figure 5" class="xfigimg" src="/files/ftp_upload/58930/58930fig5.jpg" /> 
Figure 5:&#160;Cytokine production by spleen cells immunized with FHA, Alum/FHA, BcfA/FHA, and Alum/BcfA/FHA. Dissociated cells were cultured with FHA for 7 days. Supernatants were tested by sandwich ELISA for (A) IFN-&#947;, (B) IL-17, and (C) IL-5. Errors bars are expressed as standard deviation of the mean of each group, n = 5 per group. ***p &#60; 0.001. The figure is adapted from a previous publication10. <a href="https://www-jove-com.remotexs.ntu.edu.sg/files/ftp_upload/58930/58930fig5large.jpg" target="_blank">Please click here to view a larger version of this figure.</a>
<img alt="Figure 6" class="xfigimg" src="/files/ftp_upload/58930/58930fig6.jpg" /> 
Figure 6:&#160;B. pertussis CFUs and calculations from harvested tissue from mice. (A) B. pertussis CFUs from homogenized mouse tissue. 0.1 mL serial dilutions incubated at 37 &#176;C for 4 days. (B) Calculations based upon CFUs obtained from homogenized trachea. CFUs were multiplied by the respective dilution and then multiplied by 10 to achieve CFUs per mL. In order to obtain CFU per organ, the CFUs per mL were multiplied by the total volume in which the organ was homogenized (0.3 mL). The CFUs per organ were then log transformed. <a href="https://www-jove-com.remotexs.ntu.edu.sg/files/ftp_upload/58930/58930fig6large.jpg" target="_blank">Please click here to view a larger version of this figure.</a>

Watch this Scientific Journal Video about Evaluation of Host-Pathogen Responses and Vaccine Efficacy in Mice at JoVE.com

Evaluation of Host-Pathogen Responses and Vaccine Efficacy in Mice

Here we present an elegant protocol for in vivo evaluation of vaccine effectiveness and host immune responses. This protocol can be adapted for vaccine models that study viral, bacterial, or parasitic pathogens.

Vaccines are a 20th century medical marvel. They have dramatically reduced the morbidity and mortality caused by infectious diseases and contributed to a ...

evaluation-of-host-pathogen-responses-and-vaccine-efficacy-in-mice

Nanyang Technological University

Research

JoVE Journal

Immunology and Infection

10.5K Views.  The Ohio State University. Here we present an elegant protocol for in vivo evaluation of vaccine effectiveness and host immune responses. This protocol can be adapted for vaccine models that study viral, bacterial, or parasitic pathogens.

Video: Evaluation of Host-Pathogen Responses and Vaccine Efficacy in Mice

Use of an Optical Trap for Study of Host-Pathogen Interactions for Dynamic Live Cell Imaging

Dynamic live cell imaging allows direct visualization of real-time interactions between cells of the immune system1, 2; however, the lack of spatial and temporal control between the phagocytic cell and microbe has rendered focused observations into the initial interactions of host response to pathogens difficult. Historically, intercellular contact events such as phagocytosis3 have been imaged by mixing two cell types, and then continuously scanning the field-of-view to find serendipitous intercellular contacts at the appropriate stage of interaction. The stochastic nature of these events renders this process tedious, and it is difficult to observe early or fleeting events in cell-cell contact by this approach. This method requires finding cell pairs that are on the verge of contact, and observing them until they consummate their contact, or do not. To address these limitations, we use optical trapping as a non-invasive, non-destructive, but fast and effective method to position cells in culture.
Optical traps, or optical tweezers, are increasingly utilized in biological research to capture and physically manipulate cells and other micron-sized particles in three dimensions4. Radiation pressure was first observed and applied to optical tweezer systems in 19705, 6, and was first used to control biological specimens in 19877. Since then, optical tweezers have matured into a technology to probe a variety of biological phenomena8-13.
We describe a method14 that advances live cell imaging by integrating an optical trap with spinning disk confocal microscopy with temperature and humidity control to provide exquisite spatial and temporal control of pathogenic organisms in a physiological environment to facilitate interactions with host cells, as determined by the operator. Live, pathogenic organisms like Candida albicans and Aspergillus fumigatus, which can cause potentially lethal, invasive infections in immunocompromised individuals15, 16 (e.g. AIDS, chemotherapy, and organ transplantation patients), were optically trapped using non-destructive laser intensities and moved adjacent to macrophages, which can phagocytose the pathogen. High resolution, transmitted light and fluorescence-based movies established the ability to observe early events of phagocytosis in living cells. To demonstrate the broad applicability in immunology, primary T-cells were also trapped and manipulated to form synapses with anti-CD3 coated microspheres in vivo, and time-lapse imaging of synapse formation was also obtained. By providing a method to exert fine spatial control of live pathogens with respect to immune cells, cellular interactions can be captured by fluorescence microscopy with minimal perturbation to cells and can yield powerful insight into early responses of innate and adaptive immunity.

A method is described to individually select, manipulate, and image live pathogens using an optical trap coupled to a spinning disk microscope. The optical trap provides spatial and temporal control of organisms and places them adjacent to host cells. Fluorescence microscopy captures dynamic intercellular interactions with minimal perturbation to cells.

Dynamic live cell imaging allows direct visualization of real-time interactions between cells of the immune system1, 2; however, the lack of spatial and ...

Use of the EpiAirway Model for Characterizing Long-term Host-pathogen Interactions

Nontypeable Haemophilus influenzae (NTHi) are human-adapted Gram-negative bacteria that can cause recurrent and chronic infections of the respiratory mucosa 1; 2. To study the mechanisms by which these organisms survive on and inside respiratory tissues, a model in which successful long-term co-culture of bacteria and human cells can be performed is required. We use primary human respiratory epithelial tissues raised to the air-liquid interface, the EpiAirway model (MatTek, Ashland, MA). These are non-immortalized, well-differentiated, 3-dimensional tissues that contain tight junctions, ciliated and nonciliated cells, goblet cells that produce mucin, and retain the ability to produce cytokines in response to infection. 
This biologically relevant in vitro model of the human upper airway can be used in a number of ways; the overall goal of this method is to perform long-term co-culture of EpiAirway tissues with NTHi and quantitate cell-associated and internalized bacteria over time. As well, mucin production and the cytokine profile of the infected co-cultures can be determined. This approach improves upon existing methods in that many current protocols use submerged monolayer or Transwell cultures of human cells, which are not capable of supporting bacterial infections over extended periods3. For example, if an organism can replicate in the overlying media, this can result in unacceptable levels of cytotoxicity and loss of host cells, arresting the experiment. The EpiAirway model allows characterization of long-term host-pathogen interactions. Further, since the source for the EpiAirway is normal human tracheo-bronchial cells rather than an immortalized line, each is an excellent representation of actual human upper respiratory tract tissue, both in structure and in function4.
For this method, the EpiAirway tissues are weaned off of anti-microbial and anti-fungal compounds for 2 days prior to delivery, and all procedures are performed under antibiotic-free conditions. This necessitates special considerations, since both bacteria and primary human tissues are used in the same biosafety cabinet, and are co-cultured for extended periods.

This method allows characterization of extended bacterial co-culture with EpiAirways, primary human respiratory epithelial tissue grown at the air-liquid interface, a biologically relevant in vitro model. The approach can be used with any microbe that is amenable to long-term co-culture.

Nontypeable Haemophilus influenzae  (NTHi) are human-adapted Gram-negative bacteria that can cause recurrent and chronic infections of the respiratory ...

A Comparative Approach to Characterize the Landscape of Host-Pathogen Protein-Protein Interactions

Significant efforts were gathered to generate large-scale comprehensive protein-protein interaction network maps. This is instrumental to understand the pathogen-host relationships and was essentially performed by genetic screenings in yeast two-hybrid systems. The recent improvement of protein-protein interaction detection by a Gaussia luciferase-based fragment complementation assay now offers the opportunity to develop integrative comparative interactomic approaches necessary to rigorously compare interaction profiles of proteins from different pathogen strain variants against a common set of cellular factors.
This paper specifically focuses on the utility of combining two orthogonal methods to generate protein-protein interaction datasets: yeast two-hybrid (Y2H) and a new assay, high-throughput Gaussia princeps protein complementation assay (HT-GPCA) performed in mammalian cells.
A large-scale identification of cellular partners of a pathogen protein is performed by mating-based yeast two-hybrid screenings of cDNA libraries using multiple pathogen strain variants. A subset of interacting partners selected on a high-confidence statistical scoring is further validated in mammalian cells for pair-wise interactions with the whole set of pathogen variants proteins using HT-GPCA. This combination of two complementary methods improves the robustness of the interaction dataset, and allows the performance of a stringent comparative interaction analysis. Such comparative interactomics constitute a reliable and powerful strategy to decipher any pathogen-host interplays.

This article focuses on the identification of high-confident interaction datasets between host and pathogen proteins using a combination of two orthogonal methods: yeast two-hybrid followed by a high-throughput interaction assay in mammalian cells called HT-GPCA.

Significant efforts were gathered to generate  large-scale comprehensive protein-protein interaction network maps. This is  instrumental to understand the ...

Isolation of Salmonella typhimurium-containing Phagosomes from Macrophages

Salmonella typhimurium is a facultative intracellular bacterium that causes gastroenteritis in humans. After invasion of the lamina propria, S. typhimurium bacteria are quickly detected and phagocytized by macrophages, and contained in vesicles known as phagosomes in order to be degraded. Isolation of S. typhimurium-containing phagosomes have been widely used to study how S. typhimurium infection alters the process of phagosome maturation to prevent bacterial degradation. Classically, the isolation of bacteria-containing phagosomes has been performed by sucrose gradient centrifugation. However, this process is time-consuming, and requires specialized equipment and a certain degree of dexterity. Described here is a simple and quick method for the isolation of S. typhimurium-containing phagosomes from macrophages by coating the bacteria with biotin-streptavidin-conjugated magnetic beads. Phagosomes obtained by this method can be suspended in any buffer of choice, allowing the utilization of isolated phagosomes for a broad range of assays, such as protein, metabolite, and lipid analysis. In summary, this method for the isolation of S. typhimurium-containing phagosomes is specific, efficient, rapid, requires minimum equipment, and is more versatile than the classical method of isolation by sucrose gradient-ultracentrifugation.

Isolation of Salmonella typhimurium-containing Phagosomes from Macrophages

We describe here a simple and quick method for the isolation of Salmonella typhimurium-containing phagosomes from macrophages by coating the bacteria with biotin and streptavidin.

Salmonella typhimurium is a facultative intracellular bacterium that causes gastroenteritis in humans. After invasion of the lamina propria, S. ...

A Suction Blister Protocol to Study Human T-cell Recall Responses In Vivo

Cutaneous antigen-recall models allow for studies of human memory responses in vivo. When combined with skin suction blister (SB) induction, this model offers accessibility to rare populations of antigen-specific T-cells representative of the cellular memory response as well as the cytokine microenvironment in situ.
This report describes the practical procedure of a cutaneous recall, an SB induction, and a harvest of antigen-specific T-cells. To exemplify the method, the tuberculin skin test is used for antigenic recall in individuals who, prior to this study, underwent a Bacillus Calmette-Gu&#233;rin vaccination against an infection with Mycobacterium tuberculosis. Finally, examples of multiplex and flow cytometric analyses of SB specimens are provided, illustrating high fractions of antigen-specific polyfunctional CD4+ T-cells available by this sampling method compared with cells isolated from the blood.
The method described here is safe and minimally invasive, provides a unique opportunity to study both innate and adaptive immune responses in vivo, and may be beneficial to a broad community of researchers working with cell-mediated immunity and human memory responses, in the context of vaccine development.

A Suction Blister Protocol to Study Human T-cell Recall Responses In Vivo

Here, we provide a demonstration of the suction blister cutaneous recall model. The model allows a simple access to study human in vivo adaptive immune responses, for instance in the context of vaccine development.

Cutaneous antigen-recall models allow for studies of human memory responses in vivo. When combined with skin suction blister (SB) induction, this model ...

Characterization of Thymus-dependent and Thymus-independent Immunoglobulin Isotype Responses in Mice Using Enzyme-linked Immunosorbent Assay

Antibodies, also termed as immunoglobulins (Ig), secreted by differentiated B lymphocytes, plasmablasts/plasma cells, in humoral immunity provide a formidable defense against invading pathogens via diverse mechanisms. One major goal of vaccination is to induce protective antigen-specific antibodies to prevent life-threatening infections. Both thymus-dependent (TD) and thymus-independent (TI) antigens can elicit robust antigen-specific IgM responses and can also induce the production of isotype-switched antibodies (IgG, IgA and IgE) as well as the generation of memory B cells with the help provided by antigen presenting cells (APCs). Here, we describe a protocol to characterize TD and TI Ig isotype responses in mice using enzyme-linked immunosorbent assay (ELISA). In this protocol, TD and TI Ig responses are elicited in mice by intraperitoneal (i.p.) immunization with hapten-conjugated model antigens TNP-KLH (in alum) and TNP-polysaccharide (in PBS), respectively. To induce TD memory response, a booster immunization of TNP-KLH in alum is given at 3 weeks after the first immunization with the same antigen/adjuvant. Mouse sera are harvested at different time points before and after immunization. Total serum Ig levels and TNP-specific antibodies are subsequently quantified using Ig isotype-specific Sandwich and indirect ELISA, respectively. In order to correctly quantify the serum concentration of each Ig isotype, the samples need to be appropriately diluted to fit within the linear range of the standard curves. Using this protocol, we have consistently obtained reliable results with high specificity and sensitivity. When used in combination with other complementary methods such as flow cytometry, in vitro culture of splenic B cells and immunohistochemical staining (IHC), this protocol will allow researchers to gain a comprehensive understanding of antibody responses in a given experimental setting.

In this paper, we describe a protocol to characterize T-dependent and T-independent immunoglobulin (Ig) isotype responses in mice using ELISA. This method used alone or in combination with flow cytometry will allow researchers to identify differences in B cell-mediated Ig isotype responses in mice following T-dependent and T-independent antigen immunization.

Antibodies, also termed as immunoglobulins (Ig), secreted by differentiated B lymphocytes, plasmablasts/plasma cells, in humoral immunity provide a ...

Evaluation of Zika Virus-specific T-cell Responses in Immunoprivileged Organs of Infected Ifnar1-/- Mice

The Zika virus (ZIKV) can induce inflammation in immunoprivileged organs (e.g., the brain and testis), leading to the Guillain-Barr&#233; syndrome and damaging the testes. During an infection with the ZIKV, immune cells have been shown to infiltrate into the tissues. However, the cellular mechanisms that define the protection and/or immunopathogenesis of these immune cells during a ZIKV infection are still largely unknown. Herein, we describe methods to evaluate the virus-specific T-cell functionality in these immunoprivileged organs of ZIKV-infected mice. These methods include a) a ZIKV infection and vaccine inoculation in Ifnar1-/- mice; b) histopathology, immunofluorescence, and immunohistochemistry assays to detect the virus infection and inflammation in the brain, testes, and spleen; c) the preparation of a tetramer of ZIKV-derived T-cell epitopes; d) the detection of ZIKV-specific T cells in the monocytes isolated from the brain, testes, and spleen. Using these approaches, it is possible to detect the antigen-specific T cells that have infiltrated into the immunoprivileged organs and to evaluate the functions of these T cells during the infection: potential immune protection via virus clearance and/or immunopathogenesis to exacerbate the inflammation. These findings may also help to clarify the contribution of T cells induced by the immunization against ZIKV.

Evaluation of Zika Virus-specific T-cell Responses in Immunoprivileged Organs of Infected Ifnar1-/- Mice

A protocol to evaluate antigen-specific T-cell responses in the immunoprivileged organs of the Ifnar1-/- murine model for the Zika virus (ZIKV) infection is described. This method is pivotal for investigating the cellular mechanisms of the protection and immunopathogenesis of ZIKV vaccines and is also valuable for their efficacy evaluation.

The Zika virus (ZIKV) can induce inflammation in immunoprivileged organs (e.g., the brain and testis), leading to the Guillain-Barr&#233; syndrome and ...

Infection of Zebrafish Larvae with Aspergillus Spores for Analysis of Host-Pathogen Interactions

Invasive aspergillosis (IA) is one of&#160;the most common fungal infections among immunocompromised individuals. Despite the availability of antifungal drugs, IA can cause &#62;50% mortality in infected immunocompromised patients. It is crucial to determine both host and pathogen factors that contribute to infection susceptibility and low survival rates in infected patients in order to develop novel therapeutics. Innate immune responses play a pivotal role in recognition and clearance of Aspergillus spores, though little is known about the exact cellular and molecular mechanisms. Reliable models are required to investigate detailed mechanistic interactions between the host and pathogen. The optical clarity and genetic tractability of zebrafish larvae make them an intriguing model&#160;to study host-pathogen interactions of multiple human bacterial and fungal infections in a live and intact host. This protocol describes a larval zebrafish Aspergillus infection model. First, Aspergillus spores are isolated and injected into the zebrafish hindbrain ventricle via microinjection. Then, chemical inhibitors such as immunosuppressive drugs are added directly to the larval water. Two methods to monitor the infection in injected larvae are described, including the 1) homogenization of larvae for colony forming unit (CFU) enumeration and 2) a repeated, daily live imaging setup. Overall, these techniques can be used to mechanistically analyze the progression of Aspergillus infection in vivo and can be applied to different host backgrounds and Aspergillus strains to interrogate host-pathogen interactions.

Infection of Zebrafish Larvae with Aspergillus Spores for Analysis of Host-Pathogen Interactions

This protocol describes an Aspergillus infection model in zebrafish larvae. Aspergillus spores are microinjected into the hindbrain of larvae, and chemical treatment is used to induce immunosuppression. Infection progression is monitored via a daily imaging setup to monitor fungal growth and immune responses as well as enumeration of live spores by colony forming unit plating.

Invasive aspergillosis (IA) is one of&#160;the most common fungal infections among immunocompromised individuals. Despite the availability of antifungal ...

Label-Free Quantitative Proteomics Workflow for Discovery-Driven Host-Pathogen Interactions

The technological achievements of mass spectrometry (MS)-based quantitative proteomics opens many undiscovered avenues for analyzing an organism&#8217;s global proteome under varying conditions. This powerful strategy applied to the interactions of microbial pathogens with the desired host comprehensively characterizes both perspectives towards infection. Herein, the workflow describes label-free quantification (LFQ) of the infectome of Cryptococcus neoformans, a fungal facultative intracellular pathogen that is the causative agent of the deadly disease cryptococcosis, in the presence of immortalized macrophage cells. The protocol details the proper protein preparation techniques for both pathogen and mammalian cells within a single experiment, resulting in appropriate peptide submission for liquid-chromatography (LC)-MS/MS analysis. The high throughput generic nature of LFQ allows a wide dynamic range of protein identification and quantification, as well as transferability to any host-pathogen infection setting, maintaining extreme sensitivity. The method is optimized to catalogue extensive, unbiased protein abundance profiles of a pathogen within infection-mimicking conditions. Specifically, the method demonstrated here provides essential information on C. neoformans pathogenesis, such as protein production necessary for virulence and identifies critical host proteins responding to microbial invasion.

Here, we present a protocol to profile the interplay between host and pathogen during infection by mass spectrometry-based proteomics. This protocol uses label-free quantification to measure changes in protein abundance of both host (e.g., macrophages) and pathogen (e.g., Cryptococcus neoformans) in a single experiment.

The technological achievements of mass spectrometry (MS)-based quantitative proteomics opens many undiscovered avenues for analyzing an organism&#8217;s ...

Double Labeling Immunofluorescence using Antibodies from the Same Species to Study Host-Pathogen Interactions

Nowadays, it is possible to find a wide range of molecular tools available to study parasite-host cell interactions. However, some limitations exist to obtain commercial monoclonal or polyclonal antibodies that recognize specific cell structures and proteins in parasites. Besides, there are few commercial antibodies available to label trypanosomatids. Usually, polyclonal antibodies against parasites are prepared in-house and could be more challenging to use in combination with other antibodies produced in the same species. Here, the protocol demonstrates how to use polyclonal and monoclonal antibodies raised in the same species to perform double labeling immunofluorescence to study host cell and pathogen interactions. To achieve the double labeling immunofluorescence, it is crucial to incubate first the mouse polyclonal antibody and then follow the incubation with the secondary mouse IgG antibody conjugated to any fluorochrome. After that, an additional blocking step is necessary to prevent any trace of the primary antibody from being recognized by the next secondary antibody. Then, a mouse monoclonal antibody and its specific IgG subclass secondary antibody conjugated to a different fluorochrome are added to the sample at the appropriate times. Additionally, it is possible to perform triple labeling immunofluorescence using a third antibody raised in a different species. Also, structures such as nuclei and actin can be stained subsequently with their specific compounds or labels. Thus, these approaches presented here can be adjusted for any cell whose sources of primary antibodies are limited.

Here, the protocol describes how to perform double labeling immunofluorescence using primary antibodies raised in the same species to study host-pathogen interactions. Also, it can include the third antibody from a different host in this protocol. This approach can be made in any cell type and pathogens.

Nowadays, it is possible to find a wide range of molecular tools available to study parasite-host cell interactions. However, some limitations exist to ...

Evaluation of Host-Pathogen Responses and Vaccine Efficacy in Mice

Summary

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Chapters in this video

Use of an Optical Trap for Study of Host-Pathogen Interactions for Dynamic Live Cell Imaging

Use of the EpiAirway Model for Characterizing Long-term Host-pathogen Interactions

A Comparative Approach to Characterize the Landscape of Host-Pathogen Protein-Protein Interactions

Isolation of Salmonella typhimurium-containing Phagosomes from Macrophages

A Suction Blister Protocol to Study Human T-cell Recall Responses In Vivo

Characterization of Thymus-dependent and Thymus-independent Immunoglobulin Isotype Responses in Mice Using Enzyme-linked Immunosorbent Assay

Evaluation of Zika Virus-specific T-cell Responses in Immunoprivileged Organs of Infected Ifnar1^-/- Mice

Infection of Zebrafish Larvae with Aspergillus Spores for Analysis of Host-Pathogen Interactions

Label-Free Quantitative Proteomics Workflow for Discovery-Driven Host-Pathogen Interactions

Double Labeling Immunofluorescence using Antibodies from the Same Species to Study Host-Pathogen Interactions