The authors would like to acknowledge the Division of Laboratory Animal Resources at Duke University and their husbandry team for their assistance and great care provided to the animals. Additionally, the authors would like to recognize the PhotoPath team within the Department of Pathology for their assistance with the audio and video portions of the manuscript. This work was supported by discretionary research funds from the Staats laboratory.

All procedures were approved and performed in accordance with the Duke University IACUC policies.
NOTE: Materials needed are provided in the Table of Materials.
1. Rabbit Sedation and Anesthesia
<ol>
	<li>Sedate the female rabbit (sexually mature; approximately 5-30 months old) by administering acepromazine intramuscularly (IM) at a dose of 1 mg/kg. Depending on the size of the animal, use a 1 or 3 mL syringe with a 25 G needle. Epaxial muscles are the preferred site of the intramuscular injection. 
	NOTE: Acepromazine can also be administered subcutaneously, but IM is preferred by the lab, as it acts more rapidly and reduces the incidence of skin lesions.</li>
	<li>Wait 10-15 minutes to allow the acepromazine to take effect.</li>
	<li>Anesthetize the rabbit with isoflurane by placing the connected nose cone over the animal&#39;s nose. Adjust the vaporizer to up to 4% isoflurane combined with up to 4 liters/minute oxygen. Rabbits have a high aversion to isoflurane, so adequate restraint is necessary when masking the animal.</li>
	<li>Once fully anesthetized, as assessed by the pinna, pedal, and/or palpebral reflex, apply ophthalmic lubricant to each eye to prevent drying of the eyes and subsequent corneal ulceration.</li>
	<li>Continually monitor reflexes and breathing during anesthesia, and reduce the isoflurane rate to 1-2% once an adequate plane of anesthesia has been reached.</li>
</ol>
2. Intranasal immunization
<ol>
	<li>Prepare immunization solution prior to animal handling.</li>
	<li>Sedate the rabbit as described above.</li>
	<li>Once the lab member is ready to administer the vaccine and the rabbit is in an adequate plane of anesthesia, turn off the isoflurane and oxygen and remove the nose cone.</li>
	<li>Place the rabbit in dorsal recumbency, and prop the neck and head at an approximate 45&#176; angle that allows easy access and visualization of both nares by the lab member administering the vaccine.</li>
	<li>Load the pipette with no more than 100 &#181;L of the vaccine solution, and quickly administer the solution in each nostril. The pipette should be held at an approximate 45&#176; angle, angled towards the medial aspect of the nasal passage. 
	NOTE: The goal of immunization is for the solution to contact the mucosal membrane of the nares, so the tip should not be placed within the nares, as this may result in abrasion or irritation of the mucosal tissues and potentially influence the immunogenicity of the nasally-administered vaccine. The vaccine should be administered quickly and done in the same manner in the other nare.</li>
	<li>Following administration in both nasal passages, maintain the rabbit in dorsal recumbency for 30 seconds to minimize leakage of the vaccine solution. 
	NOTE: The lab will typically administer no more than 100 &#181;L per nostril at a time. If a larger volume is to be administered, with a maximum total of 500 &#181;L, the vaccine can be given in 100 &#181;L aliquots with a 30 second rest period between immunizations, and additional administrations of vaccine repeated, with 30 seconds of rest between each administration, until the total vaccine volume is delivered.</li>
	<li>Following immunization, place the rabbit on the ventrum for recovery and closely monitor the animal until it can maintain sternal recumbency.</li>
</ol>
3. Milk collection
<ol>
	<li>Sedate the lactating rabbit as described above.</li>
	<li>Clean the skin over the marginal ear vein with the alcohol swab/wipe.</li>
	<li>Using a 1 mL syringe and 25 g needle, administer approximately 1-2 IU of oxytocin intravenously via the marginal ear vein to induce milk letdown. 
	NOTE: Due to the smooth muscle relaxation, it is common for the rabbit to urinate or defecate following administration of oxytocin.</li>
	<li>Following oxytocin administration, apply pressure to the injection site with the piece of gauze.</li>
	<li>While maintaining the anesthesia mask over the rabbit&#39;s nose, prop the rabbit on its hindquarters. 
	NOTE: Milk collection can also be performed with the animal in lateral recumbency, but the lab finds that collection is easier when the rabbit is propped up on its rump with an assistant holding the rabbit upright with the anesthesia mask.</li>
	<li>Open the sterile tube to prepare for milk collection and locate the mammary tissue and associated teats. The teats are typically surrounded by wet fur from recent nursing, and the mammary tissue is easily palpable when full of milk.</li>
	<li>Grasp the mammary tissue associated with a teat between the thumb and forefinger and apply a gentle, massaging pressure to the glandular tissue in the direction of the teat. Place the collection tube over the teat to collect the expressed milk. 
	NOTE: It can sometimes take several minutes for the oxytocin to be effective, and milk production appears to vary among mammary glands. If milk expression is not successful, wait several minutes or rotate around to the additional mammary glands. Milk from all teats can be collected in the same vial. Typically, several milliliters of milk can be easily collected from a lactating doe.</li>
	<li>Following milk collection, turn the isoflurane and oxygen off, and allow the rabbit to recover while being closely monitored until the animal is able to maintain sternal recumbency.</li>
</ol>

The authors have nothing to disclose.

Although not described in the above protocol, successful breeding of the rabbits is necessary for this maternal model and to allow for milk collection. Rabbits are easily bred by live cover in a research setting. It is recommended that does be transferred to the buck&#39;s cage for breeding, as does can be territorial and aggressive if kept in their own cage with the buck. If females are non-receptive after 15 minutes (as indicated by running away biting, or vocalizing), the doe should be placed back into her own cage. T...

Studies of maternal immunization and antibody transfer are invaluable for numerous reasons, as this is the initial route of immunity transfer and subsequent protection from pathogens and diseases in newborns and infants. Maternal immunization has the potential to positively impact both maternal and infant/child health at the global level by reducing morbidity and mortality associated with certain pathogens during this vulnerable period1. The main goal of this strategy is to increase the levels of specific maternal antibodies throughout pregnancy. These antibodies can then be transferred to the newborn and infant at levels sufficient enough to protect against infections until their immune system is mature enough to adequately respond to challenges1,2,3. Previous work has demonstrated that higher antibody titers at birth are associated with either complete protection or a delayed onset and reduced severity of numerous different infectious diseases in the newborn, including tetanus, pertussis, respiratory syncytial virus (RSV), influenza, and group B streptococcal infections1,2,3.
In humans, maternal antibodies are transferred passively across the placenta and are also transferred through the breast milk via nursing. Previous work has demonstrated that HIV-specific IgA levels in human breast milk from mothers infected with the virus were associated with reduced postnatal transmission of the virus, suggesting a protective role for breast milk anti-HIV IgA4. Studies in nonhuman primates have demonstrated that immunization against HIV can induce a significant antibody response in the breast milk, and although similar serum IgG responses were induced following systemic versus mucosal immunization, mucosal immunization induced a significantly higher IgA response within the milk5,6.
Identifying a translationally appropriate animal model for these studies should take into account the placentation type and mechanisms of passive antibody transfer, as well as the transfer of antibodies through breast milk. There are three main types of placentation in mammals based on the tissue types and layers at the materno-fetal interface, including hemochorial (primates, rodents and rabbits), endotheliochorial (carnivores), and epitheliochorial (horses, pigs, and ruminants). The hemochorial placenta is the most invasive type, allowing for direct communication between the maternal blood supply and the chorion, or the outermost fetal membrane. Based on the number of trophoblast layers, there are several variations of hemochorial placentation, including the hemomonochorial placenta found in primates, the hemodichorial placenta in rabbits, and the hemotrichorial placenta observed in rats and mice7. This direct contact between maternal blood supply and chorion allows for the passive transfer of antibodies across the placenta during gestation. IgG is the only antibody class that significantly crosses the human placenta8, whereas IgA is the predominant class of Ig found in human breast milk9. Of the scientifically relevant models, only primates (including humans), rabbits, and guinea pigs transfer IgG in utero and IgA in the milk10,11. Therefore, the rabbit model incorporates factors comparable to those in humans that control transplacental transfer of IgG and lactational transfer of IgA.
In addition to serving as an exceptional model for maternal immunity and vaccine development, similarities between the rabbit and human nasal cavities make them an appropriate model for intranasal immunization. The volume of the rabbit nasal cavity is more similar to humans than rodent models based on relative body mass12. Additionally, Casteleyn et al. 12 demonstrated that the nasal associated lymphoid tissue (NALT) is more voluminous in the rabbit compared to rodents. The NALT is located primarily at the ventral and ventromedial aspect of the ventral nasal meatus and at the lateral and dorsolateral aspect of the nasopharyngeal meatus in rabbits, whereas in rodents, the lymphoid tissue is only present along the ventral aspect of the nasopharyngeal meatus12. In rabbits, the structure and location of the intraepithelial and lamina propria lymphocytes, as well as the isolated lymphoid follicles, are similar to humans12.
Additional advantages of using the rabbit as a model for maternal and mucosal immunity include their high fecundity and relatively short gestation period. Large auricular blood vessels allow for relatively easy access to large volumes of blood for serial collections. A variety of mucosal samples can be collected for antigen-specific antibody response assays, including breast milk13 (when lactating), mucosal secretions or washes (e.g., oral14,15,16, bronchoalveolar lavage13,17,18,19, vaginal20,21,22), and feces20,23,24,25. Milk samples can be easily collected during lactation to assess the presence of antigen-specific antibody responses. Though not as abundant as for humans and mice, a wide variety of experimental reagents are available for rabbit-specific studies and assays. In this article, we will describe and demonstrate intranasal immunization and milk collection in New Zealand White rabbits (Oryctolagus cuniculus).

<table border="1" fo:keep-together.within-page="1" fo:keep-with-next.within-page="always">
	<tbody>
		<tr>
			<td>Intranasal Immunization</td>
			<td></td>
			<td></td>
			<td></td>
		</tr>
		<tr>
			<td>Anesthesia Machine/Vaporizer</td>
			<td>Vet Equip</td>
			<td>901807</td>
			<td></td>
		</tr>
		<tr>
			<td>Hypodermic Needle (25 g)</td>
			<td>Terumo</td>
			<td>07-806-7584</td>
			<td></td>
		</tr>
		<tr>
			<td>Isoflurane (250 mL Bottle)</td>
			<td>Patterson Veterinary</td>
			<td>07-893-1389</td>
			<td>2-4%</td>
		</tr>
		<tr>
			<td>Luer Lock Syringe (1 mL)</td>
			<td>Air-Tite</td>
			<td>07-892-7410</td>
			<td></td>
		</tr>
		<tr>
			<td>Mucosal Vaccine</td>
			<td>N/A</td>
			<td>N/A</td>
			<td>Experimental Vaccine</td>
		</tr>
		<tr>
			<td>Nose Cone</td>
			<td>McCulloch Medical</td>
			<td>07891-1097</td>
			<td></td>
		</tr>
		<tr>
			<td>Pipette Tips</td>
			<td>VWR</td>
			<td>53503-290</td>
			<td></td>
		</tr>
		<tr>
			<td>Pipettor</td>
			<td>VWR</td>
			<td>89079-962</td>
			<td></td>
		</tr>
		<tr>
			<td>PromAce (Acepromazine maleate)</td>
			<td>Boehringer Ingelheim</td>
			<td>07-893-5734</td>
			<td>1mg/kg IM</td>
		</tr>
		<tr>
			<td>Puralube Sterile Ophthalmic Ointment</td>
			<td>Dechra</td>
			<td>07-888-2572</td>
			<td></td>
		</tr>
		<tr>
			<td>Milk Collection</td>
			<td></td>
			<td></td>
			<td></td>
		</tr>
		<tr>
			<td>Alcohol Prep 2-ply</td>
			<td>Covidien</td>
			<td>07-839-8871</td>
			<td></td>
		</tr>
		<tr>
			<td>Anesthesia Machine/Vaporizer</td>
			<td>Vet Equip</td>
			<td>901807</td>
			<td></td>
		</tr>
		<tr>
			<td>Hypodermic Needles (25 g)</td>
			<td>Terumo</td>
			<td>07-806-7584</td>
			<td></td>
		</tr>
		<tr>
			<td>Isoflurane (250 mL Bottle)</td>
			<td>Patterson Veterinary</td>
			<td>07-893-1389</td>
			<td>2-4%</td>
		</tr>
		<tr>
			<td>Luer Lock Syringe (1 mL)</td>
			<td>Air-Tite</td>
			<td>07-892-7410</td>
			<td></td>
		</tr>
		<tr>
			<td>Non-Woven Sponge (4x4)</td>
			<td>Covidien</td>
			<td>07-891-5815</td>
			<td></td>
		</tr>
		<tr>
			<td>Nose Cone</td>
			<td>McCulloch Medical</td>
			<td>07891-1097</td>
			<td></td>
		</tr>
		<tr>
			<td>PromAce (Acepromazine Maleate)</td>
			<td>Boehringer Ingelheim</td>
			<td>07-893-5734</td>
			<td>1mg/kg IM</td>
		</tr>
		<tr>
			<td>Puralube Sterile Ophthalmic Ointment</td>
			<td>Dechra</td>
			<td>07-888-2572</td>
			<td></td>
		</tr>
		<tr>
			<td>Sterile Conical Vial (15 mL)</td>
			<td>Falcon</td>
			<td>14-959-49B</td>
			<td></td>
		</tr>
	</tbody>
</table>

intranasal immunization and milk collection in studies of maternal immunization in new zealand white rabbits (oryctolagus cuniculus)

Due to similarities in placentation and antibody transfer with humans, rabbits are an excellent model of maternal immunization. Additional advantages of this research model are the ease of breeding and sample collection, relatively short gestation period, and large litter sizes. Commonly assessed routes of immunization include subcutaneous, intramuscular, intranasal, and intradermal. Nonterminal sample collection for the chronological detection of the immunologic responses to these immunizations include the collection of blood, from both dams and kits, and milk from the lactating does. In this article, we will demonstrate techniques our lab has utilized in studies of maternal immunization in New Zealand White rabbits (Oryctolagus cuniculus), including intranasal immunization and milk collection.

An overview of a typical maternal intranasal immunization study design is depicted in Figure 1, incorporating the immunizations, breeding, kindling, lactation, and antibody transfer. Though not illustrated, blood should be collected prior to the initial immunization for baseline measurements and throughout the remainder of the study at regular intervals. Blood is easily obtained via the central ear artery with mild sedation and a topical analgesic agent (e.g., lidocaine 2.5% and prilocaine 2...

Watch this Scientific Journal Video about Intranasal Immunization and Milk Collection in Studies of Maternal Immunization in New Zealand White Rabbits Oryctolagus cuniculus at JoVE.com

Intranasal Immunization and Milk Collection in Studies of Maternal Immunization in New Zealand White Rabbits Oryctolagus cuniculus

This article describes and demonstrates the administration of intranasal vaccines and the collection of milk from lactating rabbits (Oryctolagus cuniculus) as a means to assess mucosal immunity in a translationally appropriate model of maternal immunization.

Intranasal Immunization and Milk Collection in Studies of Maternal Immunization in New Zealand White Rabbits (Oryctolagus cuniculus)

Due to similarities in placentation and antibody transfer with humans, rabbits are an excellent model of maternal immunization. Additional advantages of ...

intranasal-immunization-milk-collection-studies-maternal-immunization

Research

JoVE Journal

Immunology and Infection

3.4K Views.  Duke University Medical Center. This article describes and demonstrates the administration of intranasal vaccines and the collection of milk from lactating rabbits (Oryctolagus cuniculus) as a means to assess mucosal immunity in a translationally appropriate model of maternal immunization. 

Video: Intranasal Immunization and Milk Collection in Studies of Maternal Immunization in New Zealand White Rabbits Oryctolagus cuniculus

Retroviral Transduction of T-cell Receptors in Mouse T-cells

T-cell receptors (TCRs) play a central role in the immune system. TCRs on T-cell surfaces can specifically recognize peptide antigens presented by antigen presenting cells (APCs)1. This recognition leads to the activation of T-cells and a series of functional outcomes (e.g. cytokine production, killing of the target cells). Understanding the functional role of TCRs is critical to harness the power of the immune system to treat a variety of immunology related diseases (e.g. cancer or autoimmunity).
It is convenient to study TCRs in mouse models, which can be accomplished in several ways. Making TCR transgenic mouse models is costly and time-consuming and currently there are only a limited number of them available2-4. Alternatively, mice with antigen-specific T-cells can be generated by bone marrow chimera. This method also takes several weeks and requires expertise5. Retroviral transduction of TCRs into in vitro activated mouse T-cells is a quick and relatively easy method to obtain T-cells of desired peptide-MHC specificity. Antigen-specific T-cells can be generated in one week and used in any downstream applications. Studying transduced T-cells also has direct application to human immunotherapy, as adoptive transfer of human T-cells transduced with antigen-specific TCRs is an emerging strategy for cancer treatment6.
Here we present a protocol to retrovirally transduce TCRs into in vitro activated mouse T-cells. Both human and mouse TCR genes can be used. Retroviruses carrying specific TCR genes are generated and used to infect mouse T-cells activated with anti-CD3 and anti-CD28 antibodies. After in vitro expansion, transduced T-cells are analyzed by flow cytometry.

We present a protocol to produce antigen-specific mouse T-cells using retroviral transduction

T-cell receptors (TCRs) play a central role in the immune system. TCRs on T-cell surfaces can specifically recognize peptide antigens presented by antigen ...

Imaging of HIV-1 Envelope-induced Virological Synapse and Signaling on Synthetic Lipid Bilayers

Human immunodeficiency virus type 1 (HIV-1) infection occurs most efficiently via cell to cell transmission2,10,11. This cell to cell transfer between CD4+ T cells involves the formation of a virological synapse (VS), which is an F-actin-dependent cell-cell junction formed upon the engagement of HIV-1 envelope gp120 on the infected cell with CD4 and the chemokine receptor (CKR) CCR5 or CXCR4 on the target cell 8. In addition to gp120 and its receptors, other membrane proteins, particularly the adhesion molecule LFA-1 and its ligands, the ICAM family, play a major role in VS formation and virus transmission as they are present on the surface of virus-infected donor cells and target cells, as well as on the envelope of HIV-1 virions1,4,5,6,7,13. VS formation is also accompanied by intracellular signaling events that are transduced as a result of gp120-engagement of its receptors. Indeed, we have recently showed that CD4+ T cell interaction with gp120 induces recruitment and phosphorylation of signaling molecules associated with the TCR signalosome including Lck, CD3&zeta;, ZAP70, LAT, SLP-76, Itk, and PLC&gamma;15.
In this article, we present a method to visualize supramolecular arrangement and membrane-proximal signaling events taking place during VS formation. We take advantage of the glass-supported planar bi-layer system as a reductionist model to represent the surface of HIV-infected cells bearing the viral envelope gp120 and the cellular adhesion molecule ICAM-1. The protocol describes general procedures for monitoring HIV-1 gp120-induced VS assembly and signal activation events that include i) bi-layer preparation and assembly in a flow cell, ii) injection of cells and immunofluorescence staining to detect intracellular signaling molecules on cells interacting with HIV-1 gp120 and ICAM-1 on bi-layers, iii) image acquisition by TIRF microscopy, and iv) data analysis. This system generates high-resolution images of VS interface beyond that achieved with the conventional cell-cell system as it allows detection of distinct clusters of individual molecular components of VS along with specific signaling molecules recruited to these sub-domains.

This article describes a method to visualize formation of an HIV-1 envelope-induced virological synapse on glass supported planar bilayers by total internal reflection fluorescence (TIRF) microscopy. The method can also be combined with immunofluorescence staining to detect activation and redistribution of signaling molecules that occur during HIV-1 envelope-induced virological synapse formation.

Human immunodeficiency virus type 1 (HIV-1) infection occurs most efficiently via cell to cell transmission2,10,11. This cell to cell transfer between ...

Establishing a Liquid-covered Culture of Polarized Human Airway Epithelial Calu-3 Cells to Study Host Cell Response to Respiratory Pathogens In vitro

The apical and basolateral surfaces of airway epithelial cells demonstrate directional responses to pathogen exposure in vivo. Thus, ideal in vitro models for examining cellular responses to respiratory pathogens polarize, forming apical and basolateral surfaces. One such model is differentiated normal human bronchial epithelial cells (NHBE). However, this system requires lung tissue samples, expertise isolating and culturing epithelial cells from tissue, and time to generate an air-liquid interface culture.
Calu-3 cells, derived from a human bronchial adenocarcinoma, are an alternative model for examining the response of proximal airway epithelial cells to respiratory insult1, pharmacological compounds2-6, and bacterial7-9 and viral pathogens, including influenza virus, rhinovirus and severe acute respiratory syndrome - associated coronavirus10-14. Recently, we demonstrated that Calu-3 cells are susceptible to respiratory syncytial virus (RSV) infection in a manner consistent with NHBE15,16 . Here, we detail the establishment of a polarized, liquid-covered culture (LCC) of Calu-3 cells, focusing on the technical details of growing and culturing Calu-3 cells, maintaining cells that have been cultured into LCC, and we present the method for performing respiratory virus infection of polarized Calu-3 cells.
To consistently obtain polarized Calu-3 LCC, Calu-3 cells must be carefully subcultured before culturing in Transwell inserts. Calu-3 monolayer cultures should remain below 90% confluence, should be subcultured fewer than 10 times from frozen stock, and should regularly be supplied with fresh medium. Once cultured in Transwells, Calu-3 LCC must be handled with care. Irregular media changes and mechanical or physical disruption of the cell layers or plates negatively impact polarization for several hours or days. Polarization is monitored by evaluating trans-epithelial electrical resistance (TEER) and is verified by evaluating the passive equilibration of sodium fluorescein between the apical and basolateral compartments17,18 . Once TEER plateaus at or above 1,000 &Omega;&times;cm2, Calu-3 LCC are ready to use to examine cellular responses to respiratory pathogens.

Establishing a Liquid-covered Culture of Polarized Human Airway Epithelial Calu-3 Cells to Study Host Cell Response to Respiratory Pathogens In vitro

The findings and conclusions in this report are those of the authors and do not necessarily represent the views of the Centers for Disease Control and Prevention.

The  apical and basolateral surfaces of airway epithelial cells demonstrate  directional responses to pathogen exposure in  vivo. Thus, ideal in vitro ...

Monitoring Changes in Membrane Polarity, Membrane Integrity, and Intracellular Ion Concentrations in Streptococcus pneumoniae Using Fluorescent Dyes

Membrane depolarization and ion fluxes are events that have been studied extensively in biological systems due to their ability to profoundly impact cellular functions, including energetics and signal transductions. While both fluorescent and electrophysiological methods, including electrode usage and patch-clamping, have been well developed for measuring these events in eukaryotic cells, methodology for measuring similar events in microorganisms have proven more challenging to develop given their small size in combination with the more complex outer surface of bacteria shielding the membrane. During our studies of death-initiation in Streptococcus pneumoniae (pneumococcus), we wanted to elucidate the role of membrane events, including changes in polarity, integrity, and intracellular ion concentrations. Searching the literature, we found that very few studies exist. Other investigators had monitored radioisotope uptake or equilibrium to measure ion fluxes and membrane potential and a limited number of studies, mostly in Gram-negative organisms, had seen some success using carbocyanine or oxonol fluorescent dyes to measure membrane potential, or loading bacteria with cell-permeant acetoxymethyl (AM) ester versions of ion-sensitive fluorescent indicator dyes. We therefore established and optimized protocols for measuring membrane potential, rupture, and ion-transport in the Gram-positive organism S. pneumoniae. We developed protocols using the bis-oxonol dye DiBAC4(3) and the cell-impermeant dye propidium iodide to measure membrane depolarization and rupture, respectively, as well as methods to optimally load the pneumococci with the AM esters of the ratiometric dyes Fura-2, PBFI, and BCECF to detect changes in intracellular concentrations of Ca2+, K+, and H+, respectively, using a fluorescence-detection plate reader. These protocols are the first of their kind for the pneumococcus and the majority of these dyes have not been used in any other bacterial species. Though our protocols have been optimized for S. pneumoniae, we believe these approaches should form an excellent starting-point for similar studies in other bacterial species.

Monitoring Changes in Membrane Polarity, Membrane Integrity, and Intracellular Ion Concentrations in Streptococcus pneumoniae Using Fluorescent Dyes

Unlike that seen for eukaryotes, there is a paucity of studies that detail membrane depolarization and ion concentration changes in bacteria, primarily as their small size makes conventional methods of measurement difficult. Here, we detail protocols for monitoring such events in the significant Gram-positive pathogen Streptococcus pneumoniae utilizing fluorescence techniques.

Membrane depolarization and ion fluxes are events that have been studied extensively in biological systems due to their ability to profoundly impact ...

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 ...

Myeloid Cell Isolation from Mouse Skin and Draining Lymph Node Following Intradermal Immunization with Live Attenuated Plasmodium Sporozoites

Malaria infection begins when the sporozoite stage of Plasmodium is inoculated into the skin of a mammalian host through a mosquito bite. The highly motile parasite not only reaches the liver to invade hepatocytes and transform into erythrocyte-infective form. It also migrates into the skin and to the proximal lymph node draining the injection site, where it can be recognized and degraded by resident and/or recruited myeloid cells. Intravital imaging reported the early recruitment of brightly fluorescent Lys-GFP positive leukocytes in the skin and the interactions between sporozoites and CD11c+ cells in the draining lymph node. We present here an efficient procedure to recover, identify and enumerate the myeloid cell subsets that are recruited to the mouse skin and draining lymph node following intradermal injection of immunizing doses of sporozoites in a murine model. Phenotypic characterization using multi-parametric flow cytometry provides a reliable assay to assess early dynamic cellular changes during inflammatory response to Plasmodium infection.

Myeloid Cell Isolation from Mouse Skin and Draining Lymph Node Following Intradermal Immunization with Live Attenuated Plasmodium Sporozoites

We describe here a protocol for isolating myeloid cells from mouse skin and draining lymph node following intradermal injection of Plasmodium sporozoites. Flow cytometry of collected cells provides a reliable assay to characterize the skin and draining lymph node inflammatory response to the parasite.

Malaria infection begins when the sporozoite stage of Plasmodium is inoculated into the skin of a mammalian host through a mosquito bite. The highly ...

Human Placental and Decidual Organ Cultures to Study Infections at the Maternal-fetal Interface

The placenta shows a large degree of interspecies anatomic variability. To best understand biology and pathophysiology of the human placenta, it is imperative to design experiments using human cells and tissues. An advantage of organ culture is maintenance of three-dimensional (3D) structural organization and extracellular matrix. The goal of the method described here is successful establishment of ex vivo human gestational tissue organ cultures and their healthy culture maintenance for 72-96 hr. The protocol details the immediate processing of research-consented, placental and decidual specimens fresh from the operating suite. These are abundant specimens that would otherwise be discarded. Detailed instructions on the sterile collection of these samples, including morphologic details on how to select appropriate tissues to establish 3D organ cultures, is provided. Placental villous and decidual tissues are microdissected into 2-3 mm3 pieces and placed separately on matrix-lined transwell filters and cultured for several days. Villous and decidual organ cultures are well suited for the study of human host-pathogen interaction. As compared to other model organisms, these human cultures are particularly advantageous to examine mechanism of infection for pathogens that demonstrate variable patterns of host specificity. As an example, we demonstrate infection of placental and decidual organ cultures with the clinically relevant, facultative intracellular bacterial pathogen Listeria monocytogenes.

A simple method to establish primary human placental (villous) and decidual organ cultures is described. Villous and decidual organ cultures are invaluable tools for studying pathogenesis at the human maternal-fetal interface. Infection with the facultative intracellular bacterium Listeria monocytogenes is demonstrated.

The placenta shows a large degree of interspecies anatomic variability. To best understand biology and pathophysiology of the human placenta, it is ...

Noninvasive Sampling of Mucosal Lining Fluid for the Quantification of In Vivo Upper Airway Immune-mediator Levels

This protocol describes noninvasive sampling of undisturbed upper airway mucosal lining fluid. It also details the extraction procedure used prior to the analysis of immune mediators in fluid eluates for the study of the airway topical immune signature, without the need for stimulation procedures (often used by other techniques). The mucosal lining fluid is sampled on a strip of filter paper placed at the anterior part of the inferior turbinate and left for 2 min of absorption. Analytes are eluted from the filter papers, and the extracted protein-based eluates are analyzed by an electrochemiluminescence-based immunoassay, allowing for the high-sensitivity quantification of low- and high-level analytes in the same sample. We measured the in vivo levels of 20 preselected immune mediators related to specific immune signaling pathways in the upper airway mucosa, but the technique is not limited to that specific panel or sampling site. The technique was first implemented in 7-year-old children from the Copenhagen Prospective Studies on Asthma in Childhood2000 (COPSAC2000) cohort with allergic rhinitis. It was thereafter used in the longitudinal COPSAC2010 birth cohort, sampled at 1 month, 2 years, and 6 years of age and at instances of acute respiratory symptoms. We successfully obtained and analyzed samples from 620 (89%) of 700 1-month-old children; a few samples were below the assay detection limit (reported as the median (Inter-Quartile Range (IQR)). The number of samples below the detection limit (i.e.&#160;from 0 to the set point for the lower limit of detection) for each mediator was 29 (7.25 - 119.5). This technique enables the quantification of the in vivo airway mucosal immune profile from birth, can be applied longitudinally, and can be applied to studies on the effect of genetics and early-life environmental exposures, pathophysiology, endotyping, and monitoring of respiratory diseases, and development and evaluation of novel therapeutics.

Noninvasive Sampling of Mucosal Lining Fluid for the Quantification of In Vivo Upper Airway Immune-mediator Levels

This protocol describes a noninvasive technique for the sampling of undisturbed mucosal lining fluid from the upper airways. It can be used to perform the quantification of in vivo levels of protein mediators, such as cytokines and chemokines, in subjects of all ages.

This protocol describes noninvasive sampling of undisturbed upper airway mucosal lining fluid. It also details the extraction procedure used prior to the ...

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 ...

Immunization of Adult Zebrafish for the Preclinical Screening of DNA-based Vaccines

The interest in DNA-based vaccination has increased during the past two decades. DNA vaccination is based on the cloning of a sequence of a selected antigen or a combination of antigens into a plasmid, which enables a tailor-made and safe design. The administration of DNA vaccines into host cells leads to the expression of antigens that stimulate both humoral and cell-mediated immune responses. This report describes a protocol for the cloning of antigen sequences into the pCMV-EGFP plasmid, the immunization of adult zebrafish with the vaccine candidates by intramuscular microinjection, and the subsequent electroporation to improve intake. The vaccine antigens are expressed as green fluorescent protein (GFP)-fusion proteins, which allows the confirmation of the antigen expression under UV light from live fish and the quantification of expression levels of the fusion protein with ELISA, as well as their detection with a western blot analysis. The protective effect of the vaccine candidates is tested by infecting the fish with Mycobacterium marinum five weeks postvaccination, followed by the quantification of the bacteria with qPCR four weeks later. Compared to mammalian preclinical screening models, this method provides a cost-effective method for the preliminary screening of novel DNA-based vaccine candidates against a mycobacterial infection. The method can be further applied to screening DNA-based vaccines against various bacterial and viral diseases.

Here we describe a protocol for the immunization of the adult zebrafish (Danio rerio) with a DNA-based vaccine and demonstrate the validation of a successful vaccination event. This method is suitable for the preclinical screening of vaccine candidates in various infection models.

The interest in DNA-based vaccination has increased during the past two decades. DNA vaccination is based on the cloning of a sequence of a selected ...