Recombinant IgA is sufficient to prevent influenza virus transmission in guinea pigs

Abstract

A serum hemagglutination inhibition (HAI) titer of 40 or greater is thought to be associated with reduced influenza virus pathogenesis in humans and is often used as a correlate of protection in influenza vaccine studies. We have previously demonstrated that intramuscular vaccination of guinea pigs with inactivated influenza virus generates HAI titers greater than 300 but does not protect vaccinated animals from becoming infected with influenza virus by transmission from an infected cage mate. Only guinea pigs intranasally inoculated with a live influenza virus or a live attenuated virus vaccine, prior to challenge, were protected from transmission (A. C. Lowen et al., J. Virol. 83:2803-2818, 2009.). Because the serum HAI titer is mostly determined by IgG content, these results led us to speculate that prevention of viral transmission may require IgA antibodies or cellular immune responses. To evaluate this hypothesis, guinea pigs and ferrets were administered a potent, neutralizing mouse IgG monoclonal antibody, 30D1 (Ms 30D1 IgG), against the A/California/04/2009 (H1N1) virus hemagglutinin and exposed to respiratory droplets from animals infected with this virus. Even though HAI titers were greater than 160 1 day postadministration, Ms 30D1 IgG did not prevent airborne transmission to passively immunized recipient animals. In contrast, intramuscular administration of recombinant 30D1 IgA (Ms 30D1 IgA) prevented transmission to 88% of recipient guinea pigs, and Ms 30D1 IgA was detected in animal nasal washes. Ms 30D1 IgG administered intranasally also prevented transmission, suggesting the importance of mucosal immunity in preventing influenza virus transmission. Collectively, our data indicate that IgG antibodies may prevent pathogenesis associated with influenza virus infection but do not protect from virus infection by airborne transmission, while IgA antibodies are more important for preventing transmission of influenza viruses.

Fig 1

Purified Ms 30D1 IgG and recombinant 30D1 antibodies react against Cal09 HA by ELISA and show HAI activity. (A) Diagram of 30D1 IgG and the recombinant antibody constructs used in the study. Ms 30D1 IgG (B), cGP/Ms 30D1 IgG (C), Ms 30D1 IgA (D), and cGP/Ms 30D1 IgA (E) are specifically reactive against purified Cal09 HA by ELISA compared to irrelevant control antibodies. (F) Ms 30D1 IgG and recombinant 30D1 antibodies at 50 μg/ml all show similar HAI activities against two H1N1 pandemic viruses: Cal09 virus and A/Netherlands/602/2009 (Neth09).

Fig 2

Both Ms 30D1 IgG and Ms 30D1 IgA show protection when administered prior to infection, but only Ms 30D1 IgG shows protection when administered after infection. Mice were administered Ms 30D1 IgG (A and E) or Ms 30D1 IgA (B and F) 3 h prior to infection and challenged with 10 LD₅₀s of a mouse-adapted A/Netherlands/602/2009 virus (Neth09-ms). The upper panel shows the percentage of weight loss, while the lower panel shows survival curves. Both Ms 30D1 IgG and Ms 30D1 IgA show a survival benefit. Mice were challenged with 10 LD₅₀s Neth09-ms and administered Ms 30D1 IgG (C and G) or Ms 30D1 IgA (D and H) 24 h after infection. The upper panel shows the percentage of weight loss, while the lower panel shows survival curves. Only Ms 30D1 IgG treatment showed a survival benefit. ns, not significant. (I) Both Ms 30D1 IgG and Ms 30D1 IgA show HAI activity against mouse-adapted Neth09 virus (Neth09-ms) at 50 μg/ml. (J) Only prophylactic Ms 30D1 IgG (but not Ms 30D1 IgA) reduces lung viral titers 3 days postinfection. Kaplan-Meier survival curves were analyzed by using a log-rank (Mantel-Cox) test, and lung titers were analyzed using a Mann-Whitney test (**, P < 0.01). Mouse experiments were completed as two independent replicates.

Fig 3

Neither Ms 30D1 IgG nor cGP/Ms 30D1 IgG prevents transmission of Cal09 virus in guinea pigs. (A and B) A 10-mg/kg dose of Ms 30D1 IgG administered intramuscularly does not prevent transmission to treated animals and elevates the HAI titer of guinea pigs above 128 by day 1. (C) No Ms 30D1 IgG is detected by Cal09 HA ELISA in the nasal washes (NW) of treated guinea pigs 2 days after administration. (D and E) A 7-mg/kg dose of cGP/Ms 30D1 IgG administered intramuscularly does not prevent transmission to treated guinea pigs and elevates the HAI titer of guinea pigs to 256 by day 1. (F) cGP/Ms 30D1 IgG delays transmission as determined by a significant difference in the nasal wash titer area under the concentration-time curve (AUC) through day 6. The AUC was analyzed using a Mann-Whitney test (*, P < 0.05). All guinea pig experiments were completed as two independent replicates.

Fig 4

Ms 30D1 IgG neutralizes lung replication in Cal09-inoculated guinea pigs and prevents transmission when instilled intranasally. (A) Cal09 infection (1,000 PFU per animal) generates greater peak nasal wash (NW) titers than lung titers in guinea pigs. (B) A 10-mg/kg intramuscular administration of Ms 30D1 IgG prior to a Cal09 1,000-PFU inoculation limits lung replication and reduces nasal wash titers only 24 h after inoculation. N.D., not determined. (C) HAI titers for antibody-treated guinea pigs inoculated with Cal09 virus in panel B. (D) Daily intranasal administration of 900 μg Ms 30D1 IgG for the first 5 days prevents transmission, while intranasal administration of 900 μg Ms Ctl IgG to 4 guinea pigs did not prevent transmission. (E and F) A single administration of 900 μg Ms 30D1 IgG (E) and a single administration of 900 ng Ms 30D1 IgG (F) both prevent transmission to treated animals. (G to I) Animals that were protected by intranasal administration of antibody did not seroconvert against Cal09 HA by ELISA; all animals treated with 900 μg of control antibody seroconverted. (J and K) Day 2 nasal washes from animals treated with 900 μg Ms 30D1 IgG daily or with a single intranasal dose of 900 μg of Ms 30D1 IgG are reactive to Cal09 HA by ELISA. Nasal wash titers were analyzed using a Mann-Whitney test (***, P < 0.001). Panels A, B, and D are representative of 2 replicates. Panels E and F are single experiments with 4 treated recipient guinea pigs per experiment.

Fig 5

Ms 30D1 IgG does not prevent transmission in ferrets but reduces alveolar wash titers. (A to F) Six ferrets were treated with 3 mg/kg of Ms 30D1 IgG intravenously, generating a serum HAI titer of 256 (B) and inoculated with 10⁶ PFU of Cal09 virus. Three animals were monitored for nasal wash titers (A) and weight loss (C), while 3 animals were sacrificed on day 3 for analysis of viral titers (D), lung lavage white blood cell count (E), and Ms 30D1 IgG lung lavage reactivity against Cal09 HA by ELISA (F). (G to I) 3 mg/kg of Ms 30D1 IgG administered intravenously did not prevent transmission to ferrets (G) and generated a serum HAI titer of 256 (H). (I) No difference in weight loss was observed between Ms 30D1 IgG-treated animals and control IgG-treated animals. The experiments in panels A, C, D, E, and F were performed with 3 treated ferrets and 3 control antibody ferrets. The experiment in panel B was performed with 6 treated ferrets and 6 control ferrets. Panels G, H, and I are representative of 2 replicates with 1 inoculated ferret, 1 Ms 30D1 IgG-treated recipient ferret, and 1 control IgG-treated recipient ferret.

Fig 6

Intramuscular injection of Ms 30D1 IgA prevents transmission of a Cal09 virus in a dose-dependent manner. (A) Guinea pigs treated intramuscularly with 1 mg/kg Ms 30D1 IgA were not protected from transmission. (B) Animals treated with 1 mg/kg Ms 30D1 IgA reached a peak serum HAI titer of 40 on day 1. (C) No Ms 30D1 IgA was detected in day 2 nasal washes by Cal09 HA ELISA. (D) Guinea pigs treated intramuscularly with 5 mg/kg Ms 30D1 IgA were protected from transmission. (E) Animals treated with 5 mg/kg Ms 30D1 IgA reached a peak serum HAI titer of 160 on day 1. (F) Ms 30D1 IgA was detected in day 2 nasal washes by Cal09 HA ELISA. Protection by the 5-mg/kg Ms 30D1 IgA treatment was analyzed by Fisher's exact test. Panels A, B, and C are representative of 2 replicates with 4 inoculated guinea pigs, 2 Ms 30D1 IgA-treated recipient guinea pigs, and 2 control IgA-treated recipient guinea pigs. Panels D, E, and F are representative of 4 replicates with 4 inoculated guinea pigs, 2 Ms 30D1 IgA-treated recipient guinea pigs, and 2 control IgA-treated recipient guinea pigs.