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

10.6K Views.  The Ohio State University. Het protocol maakt het mogelijk gastheerreacties op een verscheidenheid aan ziekteverwekkers en vaccinformuleringen te evalueren. Het belangrijkste voordeel van deze techniek is dat het mogelijk maakt de evaluatie van bacteriële last en immuunresponsen in vivo, met behulp van een kanekeurbare dier model. Het aantonen van deze procedure is Kacy Yount, een afgestudeerde student van mijn laboratorium.Voor immunisatie, bevestigen een gebrek aan reactie op teen knijpen in een verdoofde, ze...

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

The Utilization of Oropharyngeal Intratracheal PAMP Administration and Bronchoalveolar Lavage to Evaluate the Host Immune Response in Mice

The host immune response to pathogens is a complex biological process. The majority of in vivo studies classically employed to characterize host-pathogen interactions take advantage of intraperitoneal injections of select bacteria or pathogen associated molecular patterns (PAMPs) in mice. While these techniques have yielded tremendous data associated with infectious disease pathobiology, intraperitoneal injection models are not always appropriate for host-pathogen interaction studies in the lung. Utilizing an acute lung inflammation model in mice, it is possible to conduct a high resolution analysis of the host innate immune response utilizing lipopolysaccharide (LPS). Here, we describe the methods to administer LPS using nonsurgical oropharyngeal intratracheal administration, monitor clinical parameters associated with disease pathogenesis, and utilize bronchoalveolar lavage fluid to evaluate the host immune response. The techniques that are described are widely applicable for studying the host innate immune response to a diverse range of PAMPs and pathogens. Likewise, with minor modifications, these techniques can also be applied in studies evaluating allergic airway inflammation and in pharmacological applications.

The host immune response to pathogen infection is a tightly regulated process. Utilizing a lipopolysaccharide lung exposure model in mice, it is possible to conduct high resolution evaluations of the complex mechanisms associated with disease pathogenesis.

The host immune response to pathogens is a complex biological process. The majority of in vivo studies classically employed to characterize host-pathogen ...

A Mouse Model for Pathogen-induced Chronic Inflammation at Local and Systemic Sites

Chronic inflammation is a major driver of pathological tissue damage and a unifying characteristic of many chronic diseases in humans including neoplastic, autoimmune, and chronic inflammatory diseases. Emerging evidence implicates pathogen-induced chronic inflammation in the development and progression of chronic diseases with a wide variety of clinical manifestations. Due to the complex and multifactorial etiology of chronic disease, designing experiments for proof of causality and the establishment of mechanistic links is nearly impossible in humans. An advantage of using animal models is that both genetic and environmental factors that may influence the course of a particular disease can be controlled. Thus, designing relevant animal models of infection represents a key step in identifying host and pathogen specific mechanisms that contribute to chronic inflammation.
Here we describe a mouse model of pathogen-induced chronic inflammation at local and systemic sites following infection with the oral pathogen Porphyromonas gingivalis, a bacterium closely associated with human periodontal disease. Oral infection of specific-pathogen free mice induces a local inflammatory response resulting in destruction of tooth supporting alveolar bone, a hallmark of periodontal disease. In an established mouse model of atherosclerosis, infection with P. gingivalis accelerates inflammatory plaque deposition within the aortic sinus and innominate artery, accompanied by activation of the vascular endothelium, an increased immune cell infiltrate, and elevated expression of inflammatory mediators within lesions. We detail methodologies for the assessment of inflammation at local and systemic sites. The use of transgenic mice and defined bacterial mutants makes this model particularly suitable for identifying both host and microbial factors involved in the initiation, progression, and outcome of disease. Additionally, the model can be used to screen for novel therapeutic strategies, including vaccination and pharmacological intervention.

Animal models have proven to be invaluable tools in defining host and pathogen specific mechanisms that contribute to the development of chronic inflammation. Here we describe a mouse model of oral infection with the human pathogen Porphyromonas gingivalis and detail methodologies to assess the progression of inflammation at local and systemic sites.

Chronic inflammation is a major driver of pathological tissue damage and a unifying characteristic of many chronic diseases in humans including ...

Intranasal Administration of Recombinant Influenza Vaccines in Chimeric Mouse Models to Study Mucosal Immunity

Vaccines are one of the greatest achievements of mankind, and have saved millions of lives over the last century. Paradoxically, little is known about the physiological mechanisms that mediate immune responses to vaccines perhaps due to the overall success of vaccination, which has reduced interest into the molecular and physiological mechanisms of vaccine immunity. However, several important human pathogens including influenza virus still pose a challenge for vaccination, and may benefit from immune-based strategies. 
Although influenza reverse genetics has been successfully applied to the generation of live-attenuated influenza vaccines (LAIVs), the addition of molecular tools in vaccine preparations such as tracer components to follow up the kinetics of vaccination in vivo, has not been addressed. In addition, the recent generation of mouse models that allow specific depletion of leukocytes during kinetic studies has opened a window of opportunity to understand the basic immune mechanisms underlying vaccine-elicited protection. Here, we describe how the combination of reverse genetics and chimeric mouse models may help to provide new insights into how vaccines work at physiological and molecular levels, using as example a recombinant, cold-adapted, live-attenuated influenza vaccine (LAIV). We utilized laboratory-generated LAIVs harboring cell tracers as well as competitive bone marrow chimeras (BMCs) to determine the early kinetics of vaccine immunity and the main physiological mechanisms responsible for the initiation of vaccine-specific adaptive immunity. In addition, we show how this technique may facilitate gene function studies in single animals during immune responses to vaccines. We propose that this technique can be applied to improve current prophylactic strategies against pathogens for which urgent medical countermeasures are needed, for example influenza, HIV, Plasmodium, and hemorrhagic fever viruses such as Ebola virus.

There is an overall lack of knowledge about how vaccines work. Here we propose the combined use of reverse genetics and bone marrow chimeric mice to gain insight into the early host immune responses to vaccines with a special focus on dendritic cells and T cell immunity.

Vaccines are one of the greatest achievements of mankind, and have saved millions of lives over the last century. Paradoxically, little is known about the ...

Protective Efficacy and Pulmonary Immune Response Following Subcutaneous and Intranasal BCG Administration in Mice

Despite global coverage of intradermal BCG vaccination, tuberculosis remains one of the most prevalent infectious diseases in the world. Preclinical data have encouraged pulmonary tuberculosis vaccines as a promising strategy to prevent pulmonary disease, which is responsible for transmission. In this work, we describe the methodology used to demonstrate in the mouse model the benefits of intranasal BCG vaccination when compared to subcutaneous. Our data revealed greater protective efficacy following intranasal BCG administration. In addition, our results indicate that pulmonary vaccination triggers a higher immune response in lungs, including Th1 and Th17 responses, as well as an increase of immunoglobulin A (IgA) concentration in respiratory airways. Our data show correlation between protective efficacy and the presence of IL17-producing cells in lungs post-Mycobacterium tuberculosis challenge, suggesting a role for this cytokine in the protective response conferred by pulmonary vaccination. Finally, we detail the global workflow we have developed to study respiratory vaccination in the mouse model, which could be extrapolated to other tuberculosis vaccines, apart from BCG, targeting the mucosal response or other pulmonary routes of administration such as the intratracheal or aerosol.

We herein detail the methodology followed to compare protective efficacy and lung immune response induced by intranasal and subcutaneous immunization with BCG in mouse model. Our results show the benefits of pulmonary vaccination and suggest a role for IL17-mediated response in vaccine-induced protection.

Despite global coverage of intradermal BCG vaccination, tuberculosis remains one of the most prevalent infectious diseases in the world. Preclinical data ...

Influenza A Virus Studies in a Mouse Model of Infection

Influenza viruses cause over 500,000 deaths worldwide1 and are associated with an annual cost of 12 - 14 billion USD in the United States alone considering direct medical and hospitalization expenses and work absenteeism2. Animal models are crucial in Influenza A virus (IAV) studies to evaluate viral pathogenesis, host-pathogen interactions, immune responses, and the efficacy of current and/or novel vaccine approaches as well as antivirals. Mice are an advantageous small animal model because their immune system is evolutionarily similar to that found in humans, they are available from commercial vendors as genetically identical subjects, there are multiple strains that can be exploited to evaluate the genetic basis of infections, and they are relatively inexpensive and easy to manipulate. To recapitulate IAV infection in humans via the airways, mice are first anesthetized prior to intranasal inoculation with infectious IAVs under proper biosafety containment. After infection, the pathogenesis of IAVs is determined by monitoring daily the morbidity (body weight loss) and mortality (survival) rate. In addition, viral pathogenesis can also be evaluated by assessing virus replication in the upper (nasal mucosa) or lower (lungs) respiratory tract of infected mice. Humoral responses upon IAV infection can be rapidly evaluated by non-invasive bleeding and secondary antibody detection assays aimed at detecting the presence of total or neutralizing antibodies. Here, we describe the common methods used to infect mice intranasally (i.n) with IAV and evaluate pathogenesis, humoral immune responses and protection efficacy.

Influenza A viruses (IAVs) are important human respiratory pathogens. To understand the pathogenicity of IAVs and to perform preclinical testing of novel vaccine approaches, animal models mimicking human physiology are required. Here, we describe techniques to evaluate IAV pathogenesis, humoral responses and vaccine efficacy using a mouse model of infection.

Influenza viruses cause over 500,000 deaths worldwide1 and are associated with an annual cost of 12 - 14 billion USD in the United States alone ...

Using a Bacterial Pathogen to Probe for Cellular and Organismic-level Host Responses

There are a variety of strategies bacterial pathogens employ to survive and proliferate once inside the eukaryotic cell. The so-called 'cytosolic' pathogens (Listeria monocytogenes, Shigella flexneri, Burkholderia pseudomallei, Francisella tularensis, and Rickettsia spp.) gain access to the infected cell cytosol by physically and enzymatically degrading the primary vacuolar membrane. Once in the cytosol, these pathogens both proliferate as well as generate sufficient mechanical forces to penetrate the plasma membrane of the host cell in order to infect new cells. Here, we show how this terminal step of the cellular infection cycle of L. monocytogenes (Lm) can be quantified by both colony-forming unit assays and flow cytometry and give examples of how both pathogen- and host-encoded factors impact this process. We also show a close correspondence of Lm infection dynamics of cultured cells infected in vitro and those of hepatic cells derived from mice infected in vivo. These function-based assays are relatively simple and can be readily scaled up for discovery-based high-throughput screens for modulators of eukaryotic cell function.

We describe both in vitro and in vivo infection assays that can be used to analyze the activities of host-encoding factors.

There are a variety of strategies bacterial pathogens employ to survive and proliferate once inside the eukaryotic cell. The so-called 'cytosolic' ...

Evaluation of T Follicular Helper Cells and Germinal Center Response During Influenza A Virus Infection in Mice

T Follicular Helper (Tfh) cells are an independent CD4+&#160;T cell subset specialized in providing help for germinal center (GC) development and generation of high-affinity antibodies. In influenza virus infection, robust Tfh and GC B cell responses are induced to facilitate effective virus eradication, which confers a qualified mouse model for Tfh-associated study. In this paper, we described protocols in detection of basic Tfh-associated immune response during influenza virus infection in mice. These protocols include: intranasal inoculation of influenza virus; flow cytometry staining and analysis of polyclonal and antigen-specific Tfh cells, GC B cells and plasma cells; immunofluorescence detection of GCs; enzyme-linked immunosorbent assay (ELISA) of influenza virus-specific antibody in serum. These assays basically quantify the differentiation and function of Tfh cells in influenza virus infection, thus providing help for studies in elucidating differentiation mechanism and manipulation strategy.

This paper describes protocols of evaluating Tfh and GC B response in mouse model of influenza virus infection.

T Follicular Helper (Tfh) cells are an independent CD4+&#160;T cell subset specialized in providing help for germinal center (GC) development and ...

Rapid In Vivo Assessment of Adjuvant's Cytotoxic T Lymphocytes Generation Capabilities for Vaccine Development

The assessment of modern sub-unit vaccines reveals that the generation of neutralizing antibodies is important but not sufficient for adjuvant selection. Therefore, adjuvants with both humoral and cellular immuno-stimulatory capabilities that are able to promote cytotoxic T lymphocytes (CTL) responses are urgently needed. Thus, faithful monitoring of adjuvant candidates that induce cross-priming and subsequently enhance CTL generation represents a crucial step in vaccine development. In here we present an application for a method that uses SIINFEKL-specific (OT-I) T cells to monitor the cross-presentation of the model antigen ovalbumin (OVA) in vivo in the presence of different adjuvant candidates. This method represents a rapid test to select adjuvants with the best cross-priming capabilities. The proliferation of CD8+ T cells is the most valuable indication of cross-priming and it is also regarded as a correlate of adjuvant-induced cross-presentation. This feature can be evaluated in different immune organs like lymph nodes and spleen. The extent of the CTL generation can also be monitored, thereby giving insights on the nature of a local (draining lymph node mainly) or a systemic response (distant lymph nodes and/or spleen). This technique further allows multiple modifications for testing drugs that can inhibit specific cross-presentation pathways and also offers the possibility to be used in different strains of conventional and genetically modified mice. In summary, the application that we present here will be useful for vaccine laboratories in industry or academia that develop or modify chemical adjuvants for vaccine research and development.

Rapid In Vivo Assessment of Adjuvant's Cytotoxic T Lymphocytes Generation Capabilities for Vaccine Development

We present here an application for a standard immunological technique (CFSE stained OT-I proliferation) intended to rapidly monitor adjuvant-mediated cytotoxic T lymphocyte (CTL) generation in vivo. This fast estimation of CTL capacities is useful for the development of prophylactic vaccines against intracellular pathogens as well as therapeutic cancer vaccines.

The assessment of modern sub-unit vaccines reveals that the generation of neutralizing antibodies is important but not sufficient for adjuvant selection. ...

Medicine

A Mouse Model for the Transition of Streptococcus pneumoniae from Colonizer to Pathogen upon Viral Co-Infection Recapitulates Age-Exacerbated Illness

Streptococcus pneumoniae (pneumococcus) is an asymptomatic colonizer of the nasopharynx in most individuals but can progress to a pulmonary and systemic pathogen upon influenza A virus (IAV) infection. Advanced age enhances host susceptibility to secondary pneumococcal pneumonia and is associated with worsened disease outcomes. The host factors driving those processes are not well defined, in part due to a lack of animal models that reproduce the transition from asymptomatic colonization to severe clinical disease. 
This paper describes a novel mouse model that recreates the transition of pneumococci from asymptomatic carriage to disease upon viral infection. In this model, mice are first intranasally inoculated with biofilm-grown pneumococci to establish asymptomatic carriage, followed by IAV infection of both the nasopharynx and lungs. This results in bacterial dissemination to the lungs, pulmonary inflammation, and obvious signs of illness that can progress to lethality. The degree of disease is dependent on the bacterial strain and host factors. 
Importantly, this model reproduces the susceptibility of aging, because compared to young mice, old mice display more severe clinical illness and succumb to disease more frequently. By separating carriage and disease into distinct steps and providing the opportunity to analyze the genetic variants of both the pathogen and the host, this S. pneumoniae/IAV co-infection model permits the detailed examination of the interactions of an important pathobiont with the host at different phases of disease progression. This model can also serve as an important tool for identifying potential therapeutic targets against secondary pneumococcal pneumonia in susceptible hosts.

A Mouse Model for the Transition of Streptococcus pneumoniae from Colonizer to Pathogen upon Viral Co-Infection Recapitulates Age-Exacerbated Illness

This paper describes a novel mouse model for the transition of pneumococcus from an asymptomatic colonizer to a disease-causing pathogen during viral infection. This model can be readily adapted to study polymicrobial and host-pathogen interactions during the different phases of disease progression and across various hosts.

Streptococcus pneumoniae (pneumococcus) is an asymptomatic colonizer of the nasopharynx in most individuals but can progress to a pulmonary and systemic ...

Evaluating the Immune Response of a Nanoemulsion Adjuvant Vaccine Against Methicillin-Resistant Staphylococcus aureus (MRSA) Infection

Nanoemulsion adjuvant vaccines have attracted extensive attention because of their small particle size, high thermal stability, and ability to induce validly immune responses. However, establishing a series of comprehensive protocols to evaluate the immune response of a novel nanoemulsion adjuvant vaccine is vital. Therefore, this article features a rigorous procedure to determine the physicochemical characteristics of a vaccine (by transmission electron microscopy [TEM], atomic force microscopy [AFM], and dynamic light scattering [DLS]), the stability of the vaccine antigen and system (by a high-speed centrifuge test, a thermodynamic stability test, SDS-PAGE, and western blot), and the specific immune response (IgG1, IgG2a, and IgG2b). Using this approach, researchers can evaluate accurately&#160;the protective effect of a novel nanoemulsion adjuvant vaccine in a lethal MRSA252 mouse model. With these protocols, the most promising nanoemulsion vaccine adjuvant in terms of effective adjuvant potential can be identified. In addition, the methods can help optimize novel vaccines for future development.

Evaluating the Immune Response of a Nanoemulsion Adjuvant Vaccine Against Methicillin-Resistant Staphylococcus aureus MRSA Infection

The present protocol prepares and evaluates the physical properties, immune response, and in vivo protective effect of a novel nanoemulsion adjuvant vaccine.

Nanoemulsion adjuvant vaccines have attracted extensive attention because of their small particle size, high thermal stability, and ability to induce ...

Evaluation of Host-Pathogen Responses and Vaccine Efficacy in Mice

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