Intralymphatic Immunotherapy and Vaccination in Mice

Summary

Prophylactic and therapeutic vaccination often fails to stimulate strong immune responses due to week drainage of the vaccine to lymph nodes and consequently poor involvement of immune cells. By direct injection of vaccine to lymph nodes, so-called intralymphatic injection, vaccine efficacy can be strongly improved and vaccine doses can be reduced.

Abstract

Vaccines are typically injected subcutaneously or intramuscularly for stimulation of immune responses. The success of this requires efficient drainage of vaccine to lymph nodes where antigen presenting cells can interact with lymphocytes for generation of the wanted immune responses. The strength and the type of immune responses induced also depend on the density or frequency of interactions as well as the microenvironment, especially the content of cytokines. As only a minute fraction of peripherally injected vaccines reaches the lymph nodes, vaccinations of mice and humans were performed by direct injection of vaccine into inguinal lymph nodes, i.e. intralymphatic injection. In man, the procedure is guided by ultrasound. In mice, a small (5-10 mm) incision is made in the inguinal region of anesthetized animals, the lymph node is localized and immobilized with forceps, and a volume of 10-20 μl of the vaccine is injected under visual control. The incision is closed with a single stitch using surgical sutures. Mice were vaccinated with plasmid DNA, RNA, peptide, protein, particles, and bacteria as well as adjuvants, and strong improvement of immune responses against all type of vaccines was observed. The intralymphatic method of vaccination is especially appropriate in situations where conventional vaccination produces insufficient immunity or where the amount of available vaccine is limited.

Introduction

Vaccines are typically injected subcutaneously or intramuscularly for stimulation of immune responses. The success of this procedure requires efficient drainage of vaccine to lymph nodes where antigen presenting cells can interact with lymphocytes for generation of T- and B-cell responses. In addition, the strength and the type of immune responses induced also depend on the density or frequency of such interactions as well as the microenvironment itself, especially the content of cytokines. As only a small fraction of a vaccine injected into a peripheral tissue reaches a lymph node, vaccinations of mice and humans by direct injection of the vaccine into lymph nodes, t....

Protocol

1. Anesthesia of Mice

1. Prepare the anesthetics by mixing ketamine (dissociative anesthetic) and xylazine (sedative and analgesic) in buffered saline. The concentrations of ketamine and xylazine in the final solution are 12.5 and 2 mg/ml, respectively.
2. Inject the anesthetics to the mice by intraperitoneal administration using a syringe with a 25-30 G needle. Use 0.1 ml/10 g of body weight.
3. Apply an ophthalmic ointment to the mice’s eyes in order to prevent drying-out of the corneas.
4. Assure that the mouse is sufficiently anesthetized by pinching its foot or toe with the forceps. If the mouse does not react with reflexes to ....

Results

The procedure of intralymphatic injections in mice, despite the surgical nature, is straight forward and relatively fast. A trained person can perform the procedure in 3-4 min. The incision that is closed with one stitch typically heals within two days (Figure 1)

Intralymphatic vaccination or immunization has been performed with mRNA, plasmid DNA, peptides, proteins, virus and bacteria. Figure 2 illustrates the antibody production after immunization with the p.......

Discussion

Intralymphatic immunization and immunotherapy have been shown to be appropriate for stimulation of both antibody responses and T-cell responses. As demonstrated in this video-article, the intralymphatic procedure of vaccination is a quick and easy method for stimulating strong immune responses in mice. A trained surgeon can perform the procedure during 3-4 min. The session can also be shared between two surgeons where one is typically doing anesthesia and the suturing and the second surgeon is doing the incision and the .......

Materials

List of materials used in this article

Name	Company	Catalog Number	Comments
Ketamine (Ketasol-100)	Graeub AG, Switzerland		Anesthetics
Xylazine (Rompun)	Bayer, Germany		Anesthetics
Viscotears Eye-Gel	Novartis, Switzerland		To keep eyes from drying out during anesthesia.
BD Micro-Fine 0.5 ml	BD Medical, France		29 G Insulin syringes with permanently attached needles
6-0 Dermalon Monofilament Nylon	Covidien, MA, USA		For sutures (0.7 metric, 18 G, 45 cm, Blue)
Curved forceps, 4.5 in	Polymed, Switzerland		For incision and holding of lymph node
Straight surgical scissors, 4.5 in	Polymed, Switzerland		For incision
Needle holder, 5.5 in	Polymed, Switzerland		To close incision with suture