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All procedures involving animal models have been reviewed by the local institutional animal care committee and the JoVE veterinary review board.
1. Immunization of Mice
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	<li>Prepare a master vaccine mix in a sterile physiological buffer such as saline or DPS. 
	NOTE: The total volume in the mix should include 10% more than that required for immunization of the whole group. Concentrations of the vaccine composition are detailed below in steps 1.1.1 ...

evaluation of vaccine-induced immunity against bacterial infection in a mouse model

Source: Caution, K., et al. <a href="https://app.jove.com/v/58930">Evaluation of Host-Pathogen Responses and Vaccine Efficacy in Mice.</a> J. Vis. Exp. (2019).
In this video, we describe a protocol to test the efficacy of an acellular pertussis vaccine against Bordetella pertussis infection in mice. Post-vaccination, the mice were subjected to infection, and their tissue homogenate was cultured to analyze the bacterial colonies as a readout of the vaccine-induced protective immunity.

<img alt="Figure 1" src="/files/ftp_upload/21609/21609_Figure1.jpg" /> 
Figure 1: Anesthesia machine setup. Shown is an anesthetic vaporizer with an attached induction chamber and scavenger system (inside the biological safety cabinet).
<img alt="Figure 2" src="/files/ftp_upload/21609/21609_Figure2.jpg" /> 
Figure 2: Calculation of delivered intranasal inoculum. (A) Schematic of serial dilution of infection inoculum ...

Evaluation of Vaccine-Induced Immunity Against Bacterial Infection in a Mouse Model

Source: Caution, K., et al. Evaluation of Host-Pathogen Responses and Vaccine Efficacy in Mice. J. Vis. Exp. (2019).
In this video, we describe a protocol ...

To study vaccine-induced immunity, take an injection filled with an acellular pertussis vaccine. The vaccine contains antigenic proteins of a respiratory disease-causing bacteria &#8212; Bordetella pertussis, and adjuvants that extend the antigen exposure for a sustained immune response.
Inject the vaccine intramuscularly into an anesthetized mouse.
Post-injection, the resident antigen-presenting cells, APCs, capture and process the antigenic proteins into peptides, presenting them on their surface. These APCs then migrate to nearby lymph nodes and interact with T cells, initiating their activation and differentiation into effector T cells.
These effector T cells circulate and migrate to various organs, including the lungs, where they reside as resident memory T cells or TRM cells, leading to immunization.&#160;
Post-immunization, intranasally administer an antibiotic-resistant strain of live Bordetella pertussis. These bacteria enter the lung tissue and activate TRM cells. The activated TRM cells secrete inflammatory cytokines that recruit the immune cells to cause bacterial clearance.
Harvest the mouse&#39;s lung tissue and homogenize it to obtain a single-cell suspension. Spread the diluted cell suspension on an antibiotic-containing agar plate and incubate it to allow bacterial colony formation.
The tissue extract from an immunized mouse exhibits fewer colonies compared to that from a non-immunized mouse, confirming vaccine-induced immunity against Bordetella pertussis.

evaluation-vaccine-induced-immunity-against-bacterial-infection-mouse

Research

JoVE Encyclopedia of Experiments

Immunology

Immune Response

Host Defense Mechanism

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 ...

JoVE Journal

Immunology and Infection

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 ...

In Vitro Cellular Activity Evaluation of the Nanoemulsion Vaccine Adjuvant Ophiopogonin D

As a principal ingredient of vaccines, adjuvants can directly induce or enhance the powerful, widespread, innate, and adaptive immune responses associated with antigens. Ophiopogonin D (OP-D), a purified component extracted from the plant Ophiopogon japonicus, has been found to be useful as a vaccine adjuvant. The problems of the low solubility and toxicity of OP-D can be effectively overcome by using a low-energy emulsification method to prepare nanoemulsion ophiopogonin D (NOD). In this article, a series of in vitro protocols for cellular activity evaluation are examined. The cytotoxic effects of L929 were determined using a cell counting kit-8 assay. Then, the secreted cytokine levels and corresponding immune cell numbers after the stimulation and culture of splenocytes from immunized mice were detected by ELISA and ELISpot methods. In addition, the antigen uptake ability in bone marrow-derived dendritic cells (BMDCs), which were isolated from C57BL/6 mice and matured after incubation with GM-CSF plus IL-4, was observed by laser scanning confocal microscopy (CLSM). Importantly, macrophage activation was confirmed by measuring the levels of IL-1&#946;, IL-6, and tumor necrosis factor alpha (TNF-&#945;) cytokines by ELISA kits after coculturing peritoneal macrophages (PMs) from blank mice with the adjuvant for 24 h. It is hoped that this protocol will provide other researchers with direct and effective experimental approaches to evaluate the cellular response efficacies of novel vaccine adjuvants.

In Vitro Cellular Activity Evaluation of the Nanoemulsion Vaccine Adjuvant Ophiopogonin D

The protocol presents detailed methods for evaluating whether the nanoemulsion ophiopogonin D adjuvant promotes effective cellular immune responses.

As a principal ingredient of vaccines, adjuvants can directly induce or enhance the powerful, widespread, innate, and adaptive immune responses associated ...

Evaluation of Vaccine-Induced Immunity Against Bacterial Infection in a Mouse Model

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