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

All experiments with live animals were conducted following a protocol approved by The Ohio State University IACUC in accordance with IACUC guidelines. C57BL/6 mice were used in all immunizations and infections. Both male and female mice are used in each group as per NIH guidelines. The number of animals per group was determined by power calculations based on the predicted differences in outcome among experimental groups. For example, 8 mice per group will yield 80% power at &#945; = 0.05 (2-sided) for a 2-sample t</e...

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, va...

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">
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		<col />
		<col />
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	<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</stron...

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 ...

The protocol enables the evaluation of host responses to a variety of pathogens and vaccine formulations. The main advantage of this technique is that it allows the evaluation of bacterial burden and immune responses in vivo, using a tractable animal model. Demonstrating this procedure is Kacy Yount, a graduate student from my laboratory.For immunization, confirm a lack of response to toe pinch in an anesthetized, six-to-twelve-week-old mouse, and load a one milliliter insulin syringe, equipped with a 28.5 gauge needle with a 0.1 milliliter of the vaccine of interest. Holding the syringe at a 45 degree angle, with the bevel facing up, insert the needle approximately five millimeters into the deltoid of the mouse, and inject the vaccine. Keep the needle inserted in the muscle for five to ten seconds, then rotate the syringe 180 degrees, so that the bevel is facing down to create a seal, and to prevent vaccine leakage, before slowly retracting the needle from the injection site.Then return the mouse to its cage, with monitoring, until full recovery. About two weeks after boosting, infect immunized mice. Grasp an anesthetized mouse by the scruff of the dorsal thorax, behind the shoulder blades and neck, and position the mouse upright to access the nose.Using a 200 microliter pipette tip, slowly apply 20 to 25 microliters of the bacterial inoculum of interest to each nare of the animal, holding the mouse until the entire inoculum has been inhaled. Then deliver an equal volume of sterile PBS to one group of mice as a negative control. At the appropriate experimental end point, place an infected mouse ventral side up on a dissection board, and secure the limbs.Wet the body with 70%ethanol, and use forceps to clasp the skin below the abdomen in the center of the pelvis. Using sharp scissors, make a vertical cut up to the mandible, and dissect the skin from the peritoneal wall. Move the skin to the sides of the carcass to create an unobstructed field for sterile organ harvest, and grasp the intact peritoneum below the ribcage, at or near the liver.Cut the peritoneum toward the ribcage to expose the lower digestive tract, and move the colon to the left to reveal the spleen. Using curved forceps, grasp the spleen, and dissect away the connective tissue with scissors. Then place the spleen in a 15 milliliter conical tube containing 3 milliliters of RPMI, and 5%fetal bovine serum on ice.Use forceps to stabilize the ribcage at the xyphoid process, and cut open the diaphragm. The lungs should deflate, and fall towards the spine. Cut open the ribcage at the sides to remove the breastplate, and cut the pulmonary arteries and veins to isolate and remove the superior lobe of the right lung.Fix the lobe in a 15 milliliter conical tube of 10%neutral buffered formalin at room temperature for at least 24 hours, and isolate the remaining lobes of the right and left lungs. Then place lobes in a 15 milliliter conical tube, containing two milliliters of 1%casein in PBS on ice. Use a one milliliter pipetman with tip to collect the blood filling the chest cavity, and transfer the blood into a pre-labeled 1.5 milliliter microcentrifuge tube on ice.Remove the parotid, sublingual, and submaxillary glands and lymph nodes covering the trachea, and open and remove the protective membranes surrounding the trachea. Using fine forceps and scissors, carefully separate the trachea from the esophagus, and any other connective tissue from the top of the collarbones to the bottom of the mandible. Use forceps to hold the trachea, and cut the trachea at top of the clavicle.Pull down the trachea to maximize the elasticity, and cut the trachea at the bottom of the mandible, right above the larynx, isolating about one centimeter of tissue. Then place the organ in a pre-labeled 1.5 milliliter microcentrifuge tube, containing 0.3 milliliters of 1%casein in PBS on ice. Now turn the mouse for clear access to the nose, and spray the head with 70%ethanol.Manually grasp the animal directly behind the skull, and use scissors to cut away the soft flesh of the snout, starting from the bottom of the nose and moving upward. Remove the fur, skin, and whiskers from around the nose, and insert the tips of a curved pair of scissors into the nares with the curves pointed downward. Cut open the nasal passage towards the eyes, creating a V-like formation.Then use fine forceps to collect the nasal septum, and soft tissue within the formation, and place the tissue in a pre-labeled 1.5 milliliter microcentrifuge tube containing 0.3 milliliters of 1%casein in PBS, on ice. To process the lung tissue, transfer the entire lung sample tube contents into a sterile 15 milliliter Dounce homogenizer, and use a pestle to homogenize the tissue. Dissociate the sample until no large particles of tissue remain, and return the homogenized suspension to the original 15 milliliter tube.Remove a 0.3 milliliter aliquot for plating colony forming units, and collect the rest of the homogenate by centrifugation. Aliquot 0.5 milliliters of supernatant into pre-labeled microcentrifuge tubes for storage at 20 degrees Celsius until cytokine expression analysis by Eliza. Then, serially dilute 0.1 milliliter aliquots of the set aside homogenized lung suspension in 0.9 milliliters of sterile PBS per dilution, and use a sterile triangle spreader to plate 0.1 milliliter of each empirically chosen dilution onto pre-labeled 10%Bordet-Gengou plus streptomycin plates.Here, representative optical density 600 measurements and calculations to achieve a one optical density bacterial suspension, to prepare a one hundredfold diluted bacterial inoculum to be delivered to mice, are shown. After seven days of vaccine antigen stimulation, immunized spleen cells from the combination vaccine group produce interferon gamma, and IL17, while significantly down regulating IL5, promoting a T helper, one T helper 17 polarization of the immune response during a B pertussis infection. Colony forming unit enumeration of bacteria recovered from respiratory tract issues of a B pertussis infected mouse can be used to assess the protective effects of the vaccine of interest.Work as quickly and as efficiently as possible in order to avoid tissue necrosis, and store the isolated tissue on ice to preserve the viability of the recovered bacteria. ELISpot flow cytometry and DNA and RNA isolation can be performed to evaluate immune cells, analyte production, and to enable epigenetic and transcriptomic analyses. When working with infectious agents such as Bordetella pertussis, it's important to remember to wear the appropriate PPE, and work in a certified biosafety cabinet in order prevent pathogen transmission.This protocol details CFU enumeration from the respiratory tract, specifically the nasal pharynx. To address questions in the vaccine and infectious disease fields about targeting bacterial colonization in the nose.

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

Research

JoVE Journal

Immunology and Infection

10.5K visualizações.  The Ohio State University. O protocolo permite a avaliação das respostas do hospedeiro a uma variedade de patógenos e formulações de vacinas. A principal vantagem dessa técnica é que ela permite a avaliação da carga bacteriana e das respostas imunes in vivo, utilizando um modelo animal tratável. Demonstrando este procedimento está Kacy Yount, uma estudante de pós-graduação do meu laboratório.Para a imunização, confirme a falta de resposta ao beliscão do dedo do sol em um rato anestesiado de sei...