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 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.
IAVs are enveloped viruses classified in the Orthomyxoviridae family3. They contain eight single-stranded RNA molecules with negative polarity3. In humans, IAVs cause seasonal epidemics and occasional pandemics of important consequence when novel viruses are introduced in the human population4. Moreover, seasonal IAVs are highly and rapidly transmitted between humans producing an elevated economic loss worldwide every year2,5. IAV symptoms include cough, nasal congestion, fever, malaise, headache, anorexia and myalgia, but the virus can also produce a more severe disease in immunocompromised patients6. In fact, the World Health Organization (WHO) calculates that seasonal influenza viruses cause 300,000 – 500,000 deaths worldwide every year1. There are only two classes of drugs currently approved by the Food and Drug Administration (FDA) for influenza prophylaxis and treatment in humans: neuraminidase (NA) inhibitors (e.g., oseltamivir) and blockers of the M2 ion channel (e.g., amantadine); however, the emergence of drug-resistant virus variants is an increasing concern. Vaccination, therefore, remains the best medical option to protect humans against IAVs infections. To date, three types of influenza vaccines licensed by the FDA for human use are available: recombinant viral hemagglutinin (HA) protein vaccines, inactivated influenza vaccines (IIV), and live-attenuated influenza vaccines (LAIV)5,7. The three vaccines are designed to induce adaptive immune response against the viral HA protein, the major target of neutralizing antibodies against IAVs.
A validated mouse model to study IAV infection in vivo
Animal models have been used to study, among others, IAV pathogenesis8,9,10,11, viral factors that contribute to disease12 and/or viral transmission13,14, and to test the efficacy of new vaccines or antiviral drugs9,10,15. Mice (Mus musculus) are the most extensively used animal model for IAV research for several reasons: 1) the immune system is evolutionarily similar to that present in humans; 2) low cost, including animal purchase, housing and reproduction; 3) small size to easily manipulate and store; 4) minimal host variability to obtain homogeneous responses and results; 5) a large knowledge of mice biology, including genome sequence; 6) many available molecular biology and/or immunology reagents; 7) available knock out (KO) mice to study the contribution of a given host protein on viral infection; and, 8) multiple mouse strains that can be exploited to evaluate the genetic basis of infections.
There are several mouse strains currently available to study IAV in vivo. Age, immune status, sex, genetic background and mouse strain as well as routes of infection, dose and viral strains all influence the outcome of IAV infection in mice. The most common mouse strains used in IAV research are C57BL/6, BALB/C and, more recently, DBA.2 mice since they are more susceptible to IAV disease than the two former strains16,17,18,19,20. Importantly, the immune response also can be different depending on the mouse strain18,19,20. Thus, it is very important to recover all the available information about the mouse and IAV strain to choose the best option for the experiment to be conducted.
Although mouse is a good animal model of infection for in vivo studies with IAV, they have several limitations, which need to be considered in the experimental design. For instance, a major limitation of using mice for in vivo studies is that IAVs do not transmit among mice. Thus, for transmission studies, more accepted animal models (e.g., ferrets or guinea pigs) are used16,17,21. In addition, there are several differences between the manifestations of IAV in mice and humans. Unlike humans, mice do not develop fever upon IAV infection; conversely they present with hypothermia16,17. In mice, IAV replication is concentrated in the lower respiratory tract (lungs) rather than the upper airways. Thus, virulence of IAV in mice is not always correlated to that seen in humans. Altogether, because the advantages outweigh the limited disadvantages, mouse represents the first animal model used to evaluate influenza viral pathogenesis, immunogenicity and protective efficacy in vaccine and antiviral studies. Moreover, it would not be ethically acceptable to conduct studies with IAV using large animal models without previous evidence in a small animal model of IAV infection. In this manuscript, we describe how to infect mice intranasally (i.n.) with IAV, how to monitor the severity and progress of viral infection and how to carry out the experiments required to evaluate humoral immune responses and protection efficacy.
All animal protocols described here were approved by the Institutional Animal Care and Use Committee (IACUC) and the Institutional Biosafety Committee (IBC) at the University of Rochester School of Medicine and Dentistry, and comply with the recommendations in the Guide for the Care and Use of Laboratory Animals of the National Research Council 22. The facilities and programs of the Vivarium and Division of Laboratory Animal Medicine of the School of Medicine and Dentistry are accredited by AAALAC International and comply with state law, federal statute and National Institutes of Health (NIH) policy. Similar requirements should be applied at each institution to adhere to the animal protocols described in this manuscript.
1. Use of Small Vertebrate Animals
NOTE: The proper Personal Protection Equipment (PPE) is required for working with mice. Minimum requirements include disposable coveralls, shoe covers, head bonnet, mask, and gloves.
2. Biosafety
NOTE: In this report, the IAVs used to infect mice are the common mouse-adapted laboratory strain of influenza A/Puerto Rico/8/34 H1N1 (PR8 WT)22,23 and a temperature sensitive LAIV variant, PR8 LAIV8. Perform all procedures that involve IAV infections (in vitro or in vivo), cell cultures, and biological samples, in a biological safety cabinet under biosafety level (BSL)-2 conditions. Utilize other BSL suits or containment conditions if highly virulent IAV strains are used.
3. Intranasal Infection
4. Characterization of Viral Pathogenesis (Figure 1)
5. Evaluation of Humoral Immune Responses (Figure 3)
6. Evaluation of Protection Efficacy Vaccines (Figure 3)
Characterization of viral pathogenesis in mice
The pathogenesis of IAV is related to the morbidity and mortality caused by its infection. These two parameters can be evaluated in mice easily: IAV morbidity is associated with body weight loss in infected mice and the percentage of survival will indicate the mortality rate (Figure 1). The body weight and survival in IAV-infected mice are usually monitored daily for at least two weeks after infection8,9,10,29. The survival data obtained can determine the MLD50 that was calculated using the method of Reed and Muench25. Another indicator of IAV pathogenesis is related to viral replication in the lower (lungs) and in the upper (nasal mucosa) respiratory tract (Figure 1). The information obtained with the viral titer in these organs corresponds to the morbidity and mortality values, and taken together provide a good indication of viral pathogenesis. It is known that IAV replication in mouse lungs peak between days 2 and 4 post-infection (p.i.), and so we recommended recovering these organs at days 2 and 4 p.i.
Figure 1: Characterization of IAV Pathogenesis in Mice: IAV pathogenesis in mice is related to its morbidity (body weight loss) and mortality (% survival), and also to the ability of IAV to replicate in the upper (nasal mucosa) and lower (lungs) respiratory tract. Briefly, on Day 0 mice are weighed and anesthetized intraperitoneally with 2,2,2-tribromoethanol (TBE) before they are infected intranasally with IAV. From day 1 to day 14, mice are weighed daily to evaluate body weight loss (morbidity) and % survival (mortality) to calculate the mouse lethal dose 50 (MLD50). On days 2 and day 4 post-infection, mice lungs and nasal mucosa are recovered and homogenized to evaluate viral replication. Mice experiments to characterize IAV pathogenesis using PR8 WT are performed under BSL-2 conditions. Please click here to view a larger version of this figure.
In Figure 2, the data obtained in the evaluation of PR8 WT pathogenesis is represented. Four groups of 6-to-8-week-old C57BL/6 mice (n = 11) were infected i.n. with the indicated doses (10, 102, 103 and 104 FFU/mouse) of PR8 WT. The body weight and survival of 5 mice per group were measured by 14 days p.i. (Figure 2A–B). All mice immunized with 104 FFU of PR8 WT lost weight quickly (Figure 2A) and all of them died at days 5 and 6 p.i. (Figure 2B). Mice immunized with 103 FFU of PR8 WT lost body weight at later times p.i. (Figure 2A) and they all died by days 7 and 8 p.i. (Figure 2B). All mice immunized with 102 FFU of PR8 WT lost body weight (Figure 2A) but only 2 of them died at day 9 p.i. (Figure 2B). Finally, mice immunized with 10 FFU of PR8 WT did not lose weight (Figure 2A) and all of them survived infection (Figure 2B). The MLD50 of PR8 WT in this experiment calculated with the Reed and Muench method25 was 1.5 x 102 FFU (Figure 2C).
To evaluate the PR8 replication in the upper and lower respiratory tract of mice infected i.n. with the indicated doses (10, 102,103, and 104 FFU/mouse), the lungs and nasal mucosa were recovered at days 2 and 4 p.i. (n = 3). After lung homogenization, the amount of virus present in this organ was analyzed by immunofluorescence assay (Figure 2D). The viral titer detected in the lungs of the different mice groups was related with the dose used to immunize them: higher viral titers were detected in lungs of mice immunized with 104 FFU of PR8 WT at days 2 and 4 p.i., while lower viral titers were detected in the lungs of mice immunized with 10 FFU of PR8 WT.
Figure 2: Representation of the Data Obtained in the Evaluation of Viral Pathogenesis: To analyze morbidity and mortality of PR8 WT, 6-to-8-week-old female C57BL/6 mice (n = 5) were infected i.n. with the indicated number of fluorescent-forming units (FFU) of PR8 WT and then monitored daily for 2 weeks for (A) body-weight loss (morbidity) and (B) survival (mortality). Data represent the means and standard deviations of results determined for individual mice (n = 5). (C) Values of % survival evaluated over 2 weeks are used to calculate the MLD50 using the Reed and Muench method25. (D) To evaluate viral replication, 6-to-8-week-old female C57BL/6 mice (n = 6) were infected i.n. with the indicated number of FFU. Viral replication in the lungs or nasal mucosa of infected mice is usually evaluated at days 2 and 4 p.i. using an immunofocus assay. Viral titers are recorded as FFU/mL. Data represent the means and SDs of results obtained in each mouse. Dashed lines are included to indicate the limit of detection (200 FFU/mL) of the assay. Mice and tissue culture experiments to assess IAV morbidity, mortality and viral titers using PR8 were performed under BSL-2 conditions. Please click here to view a larger version of this figure.
Evaluation of the humoral responses induced after vaccination
Humoral responses induced against IAV infection play an essential role in controlling viral infection. For this reason, it is very important to analyze the humoral responses elicited by IAV WT infections or by new IAV vaccines (Figure 3). In the mouse model, 14 days after immunization, mice are bled to analyze the presence of antibodies against the total viral proteins by ELISA (Figure 4), and the presence of neutralizing antibodies targeting the viral HA protein, which is usually analyzed by common serologic approaches, such as a HAI assay (Figure 5) and/or a VNA (Figure 6).
Figure 3: Scheme of the Different Steps in the Characterization of Safety, Immunogenicity and Protection Efficacy of a LAIV: When a new LAIV is developed, the mouse model of IAV infection is usually used to test safety, immunogenicity and protection efficacy. In this scheme, the experiments to assess safety (A), immunogenicity (B) and protection efficacy (C) of a candidate LAIV are indicated. (A) To evaluate the safety of a LAIV it is necessary to assay the same parameters (morbidity, mortality and viral replication in the upper and lower respiratory tract) described in Figure 1. To evaluate immunogenicity, mice are immunized and 14 days after vaccination they are bled by submandibular puncture. Humoral responses are analyzed by assessing the presence of total antibodies against IAV proteins by enzyme-linked immunosorbent assay (ELISA) and by evaluating the presence of neutralizing antibodies by hemagglutination inhibition assay (HAI) and/or virus microneutralization assay (VNA). (C) To evaluate protection efficacy, 15 days after vaccination, mice are challenged with IAV WT. Protection efficacy is evaluated by the measurement of mice morbidity, mortality and viral titers in the lungs of challenged mice as described in Figure 1. Mice experiments to characterize safety, immunogenicity and protection efficacy of a LAIV candidate are performed under BSL-2 conditions. Other BSL suits or containment conditions may be required depending on the IAV strain used for the challenge of vaccinated mice. Please click here to view a larger version of this figure.
To assess immunogenicity of PR8 LAIV, three groups of 6-to-8-week-old C57BL/6 mice (n = 5) were immunized with different doses (102,103, and 104 FFU/mouse) of PR8 LAIV or mock vaccinated with 1x PBS as a negative control. Fourteen days after vaccination, mice sera were collected by submandibular bleeding26 (Figure 3). Antibody responses against total viral proteins were evaluated by ELISA using a 1:200 dilution of cell extract of PR8-infected MDCK cells (Figure 4). Each serum was analyzed individually. The results indicate that the sera from mice immunized with the higher dose (104 FFU) of PR8 LAIV had higher antibody titers against total PR8 proteins than the sera of mice immunized with the lower dose (102 FFU). Sera control (mock-infected mice) did not show reactivity against PR8 antigens.
Figure 4: Schematic Representation of the Enzyme-linked Immunosorbent Assay (ELISA) to Evaluate Humoral Antibody Responses: Levels of PR8-specific antibodies present in infected mice are determined by ELISA in 96-well polystyrene plates coated with cell extracts from mock (control) or PR8-infected MDCK cells. Briefly, at 14 days p.i. (Figure 3), mice immunized with the indicated doses of PR8 LAIV were bled by submandibular puncture and sera were collected. Each serum was evaluated individually by ELISA for IgG antibodies against total PR8 proteins. Graph data represent the means and standard deviations of the results obtained from individual mice sera (n = 6). O.D, optical density. ELISA assays were performed in a BSL-2 cabinet. Please click here to view a larger version of this figure.
To analyze the presence of neutralizing antibodies in the mouse serum using the HAI assay, serial 2-fold dilutions of sera (previously inactivated at 56 °C) of each group of mice immunized with different doses (102, 103, and 104 FFU) of PR8 LAIV were incubated with 4 HAU of PR8 WT for 1 h at RT (Figure 5). Then, 0.5 % of turkey RBCs were added to the PR8-sera mixtures. The upper part of Figure 5 is a schematic representation of the possible HAI results: when the serum contains neutralizing antibodies, RBCs precipitation is observed in the bottom of the well because the antibodies bound to the viral HA protein prevent the hemagglutination of the RBCs. On the other hand, in the absence of neutralizing antibodies, hemagglutination of the RBCs is observed. Then, the HAI titer is calculated as the reciprocal of the dilution of serum in the last well lacking hemagglutination. The HAI results showed in the lower part of Figure 5 indicate that the serum from mouse vaccinated with the greatest amount of PR8 LAIV (104 FFU) have the higher titer of neutralizing antibodies while the serum from a mouse immunized with the lower dose (102 FFU) have the lowest titer of neutralizing antibodies against PR8. No neutralizing antibodies were present in serum control (mock-infected mice).
Figure 5: Hemagglutination Inhibition (HAI) Assay: Levels of neutralizing antibodies against PR8 WT in infected mice can be easily evaluated by HAI assay. Briefly, 2-fold serial dilutions (starting dilution 1:10) of sera from mice immunized with the indicated doses of PR8 LAIV were mixed (1:1) with 4 HAU of PR8 WT for 1 h at RT in a 96-well V-bottom plate. After 1 h incubation, 0.5% RBCs were added to the virus-serum mixtures for 30 – 60 min on ice. HAI titers are determined by identifying the last well in which the RBCs form a red button and hemagglutination does not occur (top). HAI assays with PR8 WT were performed in a BSL-2 cabinet. Please click here to view a larger version of this figure.
In Figure 6, the data obtained in the evaluation of neutralizing antibodies in mouse sera by VNA is represented. 200 FFU of PR8 WT were mixed with serial 2-fold dilutions of sera (previously inactivated at 56 °C) from mice immunized with different doses (102, 103, and 104 FFU) of PR8 LAIV for 1 h at RT. Each serum sample was assessed in triplicate. The virus-sera mixtures were used to infect monolayers of MDCK cells in a 96-well plate. The upper part of Figure 6 is a schematic representation of the possible VNA results: absence of neutralizing antibodies in the serum results in CPE at approximately 3 – 4 days p.i. In contrast, when neutralizing antibodies are present (intact cell monolayer), they bind to the viral HA and prevent viral infection, and there is no CPE. Presence of CPE is usually visualized by staining infected cells with crystal violet, and viral neutralization titers are calculated as the reciprocal of the last dilution at which infection is completely blocked. The results of the VNA are represented in the lower part of Figure 6. The serum from mice immunized with the high amount of PR8 LAIV (104 FFU) have the greatest titer of neutralizing antibodies while sera from mice immunized with the lower dose or PR8 LAIV (102 FFU) have the lowest titer of neutralizing antibodies, similar to results obtained with the HAI assay. No neutralizing antibodies were present in the serum from mock-infected mice.
Figure 6: Virus Microneutralization Assay (VNA): An alternative to the HAI assay, the VNA assay can evaluate the presence of PR8 WT neutralizing antibodies. Briefly, sera from mice vaccinated with the indicated doses of PR8 LAIV were serially 2-fold diluted (start dilution 1:5) in 96-well plates and mixed 1:1 with 200 FFU of PR8 WT for 1 h at RT. After 1 h, the virus-serum mixtures were used to infect 96-well plates of MDCK cells. Infected cells were incubated for 3 – 4 days until complete cytopathic effect. The 96-well plates were then stained with 0.1% crystal violet. VNA titers are determined by identifying the highest dilution to retain a confluent cell monolayer. VNA with PR8 WT were performed under BSL-2 conditions. Please click here to view a larger version of this figure.
Evaluation of protection efficacy of IAV vaccines in mice
When a new LAIV is developed, its safety, immunogenicity and protection efficacy must be tested in vivo, and the mouse model is usually the first animal model chosen to analyze these parameters. Figure 3 is a scheme of the different steps needed to characterize a new LAIV in mice. To evaluate safety of a LAIV (Figure 3A), mice are infected i.n. using a similar protocol to the experimental procedures described in the pathogenesis section (section 4), and the body weight and survival monitoring will provide information related to the morbidity and mortality of the LAIV (Figures 2A–2B), including the MLD50. In addition, upon inoculation with different vaccine doses, the replication of LAIV in the lower (lungs) and in the upper (nasal mucosa) respiratory tract should be assessed8,9,10,11,29 (Figure 2D). To test immunogenicity, sera from mice vaccinated with the LAIV are collected before challenge with PR8 WT (homologous challenge), using similar protocols to those described in the immunogenicity section (section 5) (Figure 3B). The presence of total and neutralizing antibodies in the sera can be detected by ELISA and HAI and/or VNA, respectively.
Lastly, to test protection efficacy, LAIV-vaccinated mice are challenged with PR8 WT and the protection efficacy is characterized by monitoring morbidity, mortality and the presence of challenge PR8 WT in the lungs, as previously described in section 4 (Figure 3C)8,9,10,11. If the LAIV induces protection against PR8 WT challenge, mice will not lose body weight, and they will survive the lethal challenge with IAV WT. Moreover, IAV WT viral titers in the lungs will not be detected, or will be significantly reduced as compared to mock (PBS) vaccinated mice8,9,10,11.
Tissue culture media and solutions | Composition | Storage | Use | Comments |
Tissue culture media: Dulbecco’s modified Eagle’s medium (DMEM), 10 % Fetal Bovine Serum (FBS), 1% Penicillin-Streptomycin-L-glutamine (PSG) (DMEM 10 % FBS 1% PSG) | 445 ml DMEM, 50 ml of FBS and 5 ml of 100x 1% Penicillin (100 units/ml)-Streptomycin (100 µg/ml)-L-glutamine (2 mM) (PSG) | Store at 4°C | This media is used for maintenance of Madin-Darby Canine Kidney (MDCK) epithelial cells | |
Post-infection media: DMEM 0.3 % Bovine Albumin (BA), 1% PSG (DMEM 0.3 % BA 1% PSG) | 491 ml DMEM, 4.2 ml of 35 % BA and 5 ml of 100x PSG | Store at 4°C | This media is used for maintenance of MDCK cells after infection with Influenza A virus (IAV) | |
10x Phosphate buffered saline (10x PBS) | 80 g of NaCl, 2 g of KCl, 11.5 g of Na2HPO4.7H2O, 2 g of KH2PO4. Add ddH2O up to 1 L. Adjust pH to 7.3 | Store at room temperature (RT) | Sterilize by autoclave | |
1x PBS | Dilute 10x PBS 1:10 with ddH2O | Store at RT | Sterilize by autoclave | |
Infection media: 1x PBS, 0.3% BA, 1% Penicillin-Streptomycin (PS) (PBS/BA/PS) | 487 ml 1x PBS sterile, 4.2 ml of 35% BA and 5 ml of 100x 1% Penicillin (100 units/ml)-Streptomycin (100 µg/ml) (PS) | Store at 4 °C | This media is used for IAV infections | |
Fixation/permeabilization solution: 4% formaldehyde, 0.5% triton X-100 diluted in 1x PBS | 400 mL Neutral Buffered Formalin 10%, 5 ml of Triton X-100 and 595 ml of 1x PBS | Store at RT | This solution is used to fix and permeabilize MDCK cells in viral titration experiments | Prepare the solution in a fume hood to prevent exposure to formaldehyde |
Blocking solution: 2.5% Bovine Serum Albumin (BSA) in 1x PBS | 2.5 g of BSA in 97.5 mL of 1x PBS | Store at 4 °C | This solution is used as a blocking solution for immunofluorescence assays and ELISAs. | Sterilize by filtration with 0.2 µm filter. |
Antibody dilution solution (1% BSA in 1x PBS) | 1 g of BSA in 99 mL of 1x PBS | Store at 4 °C | This solution is used for the dilution of primary and secondary antibodies in immunofluorescence assays and ELISAs | Sterilize by filtration with 0.2 µm filter |
0.1% crystal violet solution | 1 g of crystal violet in 400 ml of methanol. Add 600 ml of ddH2O | Store at RT | This solution is used to fix and stain MDCK cells in viral neutralization assays | |
Tosylsulfonyl phenylalanyl chloromethyl ketone (TPCK)-treated trypsin | Prepare a 1,000x stock solution at 1 mg/ml in ddH2O | Store at -20 °C | The TPCK-trypsin is added in IAV infections | Make 100 µl aliquots |
RIPA buffer | 10 mM Tris-Cl (pH 8.0), 1 mM EDTA, 1% Triton X-100, 0.1% sodium deoxycholate, 0.1% SDS, 140 mM NaCl | Store at 4 °C | This solution is used to make cell extracts |
Table 1: Tissue Culture Media and Solutions.
The mouse model of IAV is widely used for in vivo studies of IAV pathogenesis, immunogenicity and protection efficacy. The small size of mice makes them easy to manipulate and store as compared to other animal models such as ferrets or guinea pigs. Moreover, the ease in terms of animal cost, housing and reproduction allow their use in pre-clinical vaccination tests in which large numbers of animals are needed. Notably, since mice have been used in multiple research disciplines, several molecular and immunology murine reagents are available to facilitate their use for IAV studies. Furthermore, the minimal host variability and the presence of an immune system that is evolutionarily similar to that present in humans make them a robust small animal model for IAV studies. Finally, the study of host-viral protein interactions and their contribution to viral pathogenesis are currently feasible by the access to KO mice. However, beside the several advantages of using mice for IAV studies, they are not useful for IAV transmission studies. Therefore, ferrets or guinea pigs are more suitable animal models to study IAV transmission. Clinical symptoms in mice infected with IAV usually appear 2 – 3 days post-infection, although this may vary depending on the mouse age, immune status, sex, genetic background and strain; as well as viral strain and dose used. Symptoms include anorexia, lethargy, huddling, ruffled fur, loss of body weight and death16,17.
In this manuscript, we describe how to evaluate viral pathogenesis in mice infected i.n. with IAV by monitoring body weight loss (morbidity), percentage of survival (mortality) and by assessing viral replication in the upper (nasal mucosa) and lower (lungs) respiratory tract (Figure 1 and Figure 2). The pathogenesis of IAV is a very important parameter not only in the context of new IAV strains but also in the safe implementation of LAIV vaccine candidates (Figure 3). Seasonal IAVs and IAVs that infect other mammals (like horses, dogs or pigs) do not usually produce signs of disease in mice11,27. In this case, the analysis of viral titers in the lungs or nasal mucosa of infected mice is the main parameter to evaluate viral pathogenesis of IAV strains. Once the lungs and nasal mucosa are collected, they are homogenized and the amount of virus present in these organs can be analyzed by plaque assay, immunofluorescence assay, tissue culture infectious dose 50 (TCID50) or another validated method8,29. We recommend evaluating viral titers using indirect immunofluorescence since the plaque assay requires a higher quantity of MDCK cells (6-well plate format) and, like the TCID50, more time (3 – 4 days) is required to calculate viral titers. However, to perform the indirect immunofluorescence assay, it is necessary to have: 1) primary specific antibodies against an IAV protein (it is recommended to use an antibody against the IAV nucleoprotein, NP, since it is the most abundant viral protein produced during viral infection); 2) secondary antibodies conjugated to a fluorophore; and 3) a fluorescence microscope to count fluorescent positive infected cells.
Since the immune system of mice is evolutionarily similar to the immune system of humans16 and because mice can elicit a strong and protective humoral response against IAV infection, the immunogenicity of IAVs (or vaccine candidates) can be easily evaluated by determining total (ELISA; Figure 4) and neutralizing (HAI, Figure 5) or VNA (Figures 6) antibody responses. To analyze the humoral responses after immunization, mice can be bled by different approved methods such as retro-orbital puncture, tail clip, saphenous vein puncture or submandibular puncture. We chose the submandibular puncture technique26 because the procedure does not require the use of anesthesia and allows us to obtain an acceptable volume of blood for immunological studies; as opposed to the retro-orbital puncture, where mice should be anesthetized, and with the tail clip or saphenous vein puncture, where the blood volume recovered usually is low.
In this manuscript, we described how to evaluate the presence of antibodies against total viral proteins by ELISA using virus infected cell extracts. Since IAV infection induces a protective immunity mediated, mainly, by antibodies against the viral HA (the main antigenic viral target), it is highly recommended to assess the amount of specific antibodies induced against HA by ELISA using recombinant purified HA proteins10. To analyze the presence of neutralizing antibodies, both HAI and VNA methods are accepted but both have advantages and disadvantages. The HAI is rapid (2-3 h) and approved by the Center for Disease and Control (CDC) to evaluate the presence of IAV neutralizing antibodies30. The VNA is more time consuming (3-4 days) but is more specific since it evaluates only antibodies that can neutralize viral infection. Notably, the VNA has been shown to detect stalk-reactive HA neutralizing antibodies31, as well as NA neutralizing antibodies32. Another advantage of the VNA is the assay sensitivity, since less IAV ( 200 FFU) is needed as compared to the HAU assay that requires approximately ~104-105 FFU. Moreover, the HAI assay can be problematic for detecting neutralizing antibodies against avian IAVs due to difficulty with interpreting results33,34,35. Additionally, VNA does not require the use of RBCs for the identification of influenza neutralizing antibodies.
Finally, we describe the experimental procedures to evaluate the three main aspects that have to be considered in developing new IAV vaccines (Figure 3): 1) safety: the vaccine must be safe and not produce disease; 2) immunogenicity: the vaccine must be immunogenic and induce both humoral and cellular protective responses; 3) protection efficacy: the vaccine must induce protection against challenge with WT homologous and/or heterologous viruses. The mouse model is a good choice for the first screening of a new IAV vaccine in vivo because it can evaluate diverse vaccination schemes and conditions, including use of different adjuvants, antigen/vaccine dose, administration and broad protection against challenge with homologous or heterologous IAVs strains16,17. However, before proceeding to human clinical trials, vaccine candidates should at a minimum be tested in a second in vivo model.
The authors have nothing to disclose.
Research on influenza virus in LM-S laboratory is partially funded by The New York Influenza Center of Excellence (NYICE), a member of the NIAID Centers of Excellence for Influenza Research and Surveillance (CEIRS). We thank Wendy Bates for her support in the corrections of the manuscript.
Madin-Darby Canine Kidney (MDCK) epithelial cells | ATCC | CCL-34 | |
Six- to eight-week-old female C57BL/6 mice | National Cancer Institute (NCI) | 01XBE | |
Turckey red blod cells | Biolink Inc | Store at 4°C | |
Dulbecco’s modified Eagle’s medium (DMEM) | Corning Cellgro | 15-013-CV | Store at 4°C |
Fetal Bovine Serum (FBS) | Seradigm | 1500-050 | Store at -20°C |
Penicillin/Streptomycin/L-Glutamine (PSG) 100X | Corning | 30-009-CI | Store at -20°C |
Penicillin/Streptomycin (PS) 100X | Corning | 30-00-CI | Store at -20°C |
Bovin Albumin solution (BA) | Sigma-Aldrich | A7409 | Store at 4°C |
Bovin Serum Albumin (BSA) | Sigma-Aldrich | A9647 | Store at 4°C |
Tosylsulfonyl phenylalanyl chloromethyl ketone (TPCK)-treated trypsin | Sigma-Aldrich | T8802 | Store at -20°C |
Neutral Buffered Formalin 10% | EMD | 65346-85 | Store at RT |
Triton X-100 | J.T.Baker | X198-07 | Store at RT |
Monoclonal Antibody anti-NP Influenza A Virus HB-65 | ATTC | H16-L10-4R5 | Store at -20°C |
Polyclonal rabbit anti-mouse immunoglobulins/FITC | Dako | F0261 | Store at 4°C |
ECL Anti-mouse IgG, Horseradish Peroxidase linked whole antibody | GE Healthcare | LNA931V/AG | Store at 4°C |
TMB substrate set | BioLegend | 421101 | Store at 4°C |
Vmax Kinetic plate reader | Molecular Devices | ||
Dounce Tissue Grinders | Thomas Scientific | 7722-7 | |
Receptor destroying enzyme, RDE (II) | Denka Seiken Co. | 370013 | Store at -20°C |
Crystal Violet | Fisher Scienctific | C581-100 | Store at RT |
96-well Cell Culture Plate | Greiner Bio-one | 655-180 | |
Cell Culture dishes 100mm | Greiner Bio-one | 664-160 | |
Nunc MicroWell 96-Well Microplates | Thermo Fisher Scienctific | 269620 | |
Nunc 96-Well Polystyrene Conical Bottom MicroWell Plates | Thermo Fisher Scienctific | 249570 | |
Puralub Vet Ointment | Dechra | 9N-76855 | |
Fluorescent microscope | Olympus | Olympus IX81 |