Monoclonal Antibody Therapy Protects Pharmacologically Immunosuppressed Mice from Lethal Infection with Influenza B Virus

Human influenza A and B viruses are highly contagious and cause similar illnesses and seasonal epidemics. Currently available antiviral drugs have limited efficacy in humans with compromised immune systems; therefore, alternative strategies for protection are needed. Here, we investigated whether monoclonal antibodies (MAbs) targeting hemagglutinin (HA) and/or neuraminidase (NA) proteins would protect immunosuppressed mice from severe infections with influenza B virus. Pharmacologically immunosuppressed BALB/c mice were inoculated with B/Brisbane/60/2008 (BR/08) influenza virus and were treated with a single dose of 1, 5, or 25 mg/kg of body weight per day of either an anti-HA MAb (1D2) or an anti-NA MAb (1F2) starting at 24 hours postinoculation (hpi).

ABSTRACT

Human influenza A and B viruses are highly contagious and cause similar illnesses and seasonal epidemics. Currently available antiviral drugs have limited efficacy in humans with compromised immune systems; therefore, alternative strategies for protection are needed. Here, we investigated whether monoclonal antibodies (MAbs) targeting hemagglutinin (HA) and/or neuraminidase (NA) proteins would protect immunosuppressed mice from severe infections with influenza B virus. Pharmacologically immunosuppressed BALB/c mice were inoculated with B/Brisbane/60/2008 (BR/08) influenza virus and were treated with a single dose of 1, 5, or 25 mg/kg of body weight per day of either an anti-HA MAb (1D2) or an anti-NA MAb (1F2) starting at 24 hours postinoculation (hpi). Monotherapy with 1D2 or 1F2 MAbs provided dose-dependent protection of mice, with decreased BR/08 virus replication and spread in the mouse lungs, compared with those of controls. Combination treatment with 1D2 and 1F2 provided greater protection than did monotherapy, even when started at 48 hpi. Virus spread was also efficiently restrained within the lungs, being limited to 6%, 10%, and 10% of that seen in active infection when treatment was initiated at 24, 48, and 72 hpi, respectively. In most cases, the expression of cytokines and chemokines was altered according to when treatment was initiated. Higher expression of proinflammatory IP-10 and MCP-1 in combination-treatment groups, but not in monotherapy groups, to some extent, promoted better control of virus spread within the lungs. This study demonstrates the potential value of MAb immunotherapy in treating influenza in immunocompromised hosts who are at increased risk of severe disease.

INTRODUCTION

Influenza A and B viruses are important human respiratory pathogens that cause annual epidemics and significant morbidity and mortality (1). Two genetically distinct subtypes of influenza A virus (H1N1 and H3N2) and two lineages of influenza B virus (B/Victoria/2/1987-like and B/Yamagata/16/1988-like) cocirculate globally. The burden of influenza B viruses is frequently underestimated, in part because of the predominance of influenza A viruses in most epidemic seasons. In general, the global frequency of influenza B viruses remains relatively low compared with that of influenza A viruses, but influenza B viruses can dominate in some influenza seasons, e.g., the 2017 to 2018 season in Europe (2). In the United States, surveillance studies reported that an average of 20% of laboratory-confirmed influenza cases in the period from 2010 to 2019 were caused by influenza B viruses, accounting for an average of 30% of the influenza-associated pediatric deaths in each influenza season (3, 4).

Currently, the quadrivalent influenza vaccine, which includes influenza A(H1N1) and A(H3N2) and influenza B viruses from both lineages, is the most widely used approach to reduce the influenza virus burden (5). Therapy with antiviral drugs is another approach and can play a leading role when vaccines are ineffective, in short supply, or unavailable. In addition to neuraminidase (NA) inhibitors (NAIs), the arsenal of anti-influenza drugs was recently extended with the approval in 2018 to 2019 of baloxavir marboxil (BXM), which targets the viral polymerase acidic (PA) protein (6). However, in high-risk groups, such as immunosuppressed individuals (representing nearly 3% of the United States population) (7), vaccination and antiviral agents may be less effective (8, 9). In immunosuppressed individuals, vaccines are inadequately immunogenic to seroprotect against influenza virus infection (8) and antiviral drugs inefficiently control prolonged viral shedding and virus spread to the lower respiratory tract (10). Moreover, the NAI oseltamivir is less effective for patients infected with influenza B virus than for those infected with influenza A virus (11, 12), and the susceptibility of influenza B viruses to BXM is lower than that of influenza A viruses (13). To counter prolonged viral replication, extended antiviral treatment regimens are used for immunosuppressed patients, but they increase the risk of drug-resistant variants emerging (14). The development of novel therapeutics, such as broadly reactive monoclonal antibodies (MAbs), may facilitate improved strategies for treating influenza virus infections, as evidenced by the success of the MAb-based immunotherapy (with palivizumab) developed for respiratory syncytial virus infections (15).

Two virus surface glycoproteins are the major targets of MAb development for defense against influenza, namely, hemagglutinin (HA) and NA. The immunodominant receptor-binding globular head domain of HA binds to cellular sialic acid-containing receptors and initiates HA-mediated virus-host membrane fusion (16). NA, the immunosubdominant and second most abundant protein (after HA) on the viral surface, is a receptor-destroying enzyme; it cleaves terminal sialic acid residues from glycoconjugates and facilitates the release of newly synthesized virus particles (17). Preclinical data from studies of both murine-origin and human-origin MAbs binding to HA or NA proteins of influenza A viruses showed that MAbs protected mice against weight loss and disease when administered prophylactically or therapeutically (18–20). Much less information is available for MAbs against influenza B viruses, although their cross-reactivity and broad protective effect have been observed in animal models (20–23). Our understanding of the potential of MAb-based immunotherapy in immunocompromised hosts is limited, but immunotherapy may be of value to these individuals by acting as an adjunct therapy for influenza virus infections.

We investigated the therapeutic potency of monotherapy and combination therapy with anti-HA (1D2) and anti-NA (1F2) MAbs against lethal infection with influenza B virus in pharmacologically immunosuppressed BALB/c mice. The combination of anti-HA and anti-NA MAbs was beneficial for controlling influenza infection in immunosuppressed hosts, indicating that anti-NA MAb therapy could be a valuable adjunct to the available antiviral drugs and/or anti-HA MAb therapy in influenza patients with deficient immunity.

RESULTS

Selection of 1D2 and 1F2 MAbs.

Several cross-reactive anti-HA and anti-NA MAbs that are protective against influenza B virus infection in immunocompetent animals have been reported (22, 23). We initially narrowed this list by examining the capacity of the MAbs to protect immunosuppressed mice against lethal challenge with influenza B/Brisbane/60/2008 (BR/08) virus. Mice received a 5 mg/kg of body weight dose of anti-HA MAb (1D2, anti-head, nonneutralizing [22]; 3F4, anti-head, virus neutralizing; or 4C2, anti-stalk, virus neutralizing—all broadly cross-reactive) or anti-NA MAb (1F2, anti-head, neutralizing) (23). Mice that received an isotype control MAb (anti-H6 HA, 8H9) exhibited progressive weight loss and succumbed to infection between 6 and 8 days postinoculation (dpi) (see Fig. S1A in the supplemental material). Treatment of immunosuppressed mice with the anti-HA and anti-NA MAbs provided variable protection. A single dose of 1D2, 4C2, or 1F2 protected 13%, 25%, and 50% of mice, respectively; administration of 3F4 did not provide protection. A two-dose regimen of 1D2, administered 2 days apart, was more effective at promoting the survival of immunosuppressed mice (50%). Moreover, only a two-dose regimen of 3F4 demonstrated protection (25%), whereas the survival benefit due to a two-dose regimen of 1F2 (38%) and 4C2 (25%) did not largely differ from one-dose regimens (Fig. S1B). Based on the highest level of protection obtained and our desire to examine the effect of targeting both virus surface proteins, the anti-HA 1D2 and anti-NA 1F2 MAbs were chosen for further study.

The persistence of MAb circulation in the blood is an important factor that influences its therapeutic potential, especially in an immunocompromised host who may have prolonged virus shedding. Therefore, we determined the half-life of the MAbs 1D2 and 1F2 in immunocompetent and pharmacologically immunosuppressed BALB/c mice (see Fig. S2 in the supplemental material). In immunocompetent mice, the half-lives of 1D2 and 1F2 were 3.98 and 14.91 days, respectively. In immunosuppressed mice, these half-lives were reduced to 2.46 and 5.38 days, respectively. At 1 day postadministration, MAb concentrations were higher in immunosuppressed than in immunocompetent mice. These findings suggest that the retention rates of the administered MAbs were higher in hosts with defective immune-response mechanisms but their bioavailability was consequently reduced.

Survival of BR/08 virus-infected MAb-treated immunosuppressed mice.

To define the therapeutic potential of the MAbs 1D2 and 1F2 in monotherapy and combination therapy, immunosuppressed BALB/c mice were lethally challenged with BR/08 and treated with different MAb regimens that varied with respect to the concentration and time of administration (Fig. 1). The control animals (virus-inoculated, anti-H6 HA [8H9] MAb-treated mice) succumbed to infection between 6 and 12 dpi; their mean survival was 9.1 days (Table 1). When administered in a single-dose regimen at 24 hpi, both 1D2 and 1F2 provided a dose-dependent protection of mice. Treatment of immunosuppressed mice with 5 or 25 mg/kg of 1D2 resulted in 62.5% and 87.5% survival, respectively (Fig. 1A). Better protection was achieved when 1F2 was administered, and doses of 5 and 25 mg/kg protected 100% of mice, whereas the lowest dose (1 mg/kg) promoted 87.5% survival (Fig. 1C). Repeated (two or three times) administration of 1D2 and 1F2 at 5 mg/kg afforded 75% to 100% protection, which was comparable with that achieved with the highest tested single-dose regimen (25 mg/kg). Treatment with three doses (75 mg/kg) of the NAI peramivir, a parenteral antiviral drug approved by the Food and Drug Administration for treating uncomplicated influenza virus infections in adults (24), resulted in 0% survival of animals. Thus, MAb monotherapy was more effective than the standard-of-care NAI peramivir at protecting immunosuppressed mice against lethal challenge with BR/08 virus. The anti-NA MAb 1F2 was more potent than the anti-HA MAb 1D2 for rescuing mice from severe morbidity and mortality.

FIG 1.

Survival of immunosuppressed mice lethally challenged with influenza BR/08 virus and treated with MAbs. Immunosuppressed BALB/c mice (n = 8/group) were inoculated intranasally with 10 MLD₅₀ of influenza BR/08 virus and treated with the anti-HA MAb 1D2 (A) or the anti-NA MAb 1F2 (C) or combinations of the two (B, D, E). Monotherapy with 1D2 and 1F2 was i.p. administered as a single dose (1, 5, or 25 mg/kg) or as repeated doses (5 mg/kg) beginning at 24 hpi. Combinations of 1D2 and 1F2 were i.p. administered as a single dose (10 and 1 mg/kg, respectively) at 24 (B), 48 (D), and 72 (E) hpi. The NAI peramivir was administered by i.m. injection as a three-dose regimen (75 mg/kg) at 24, 96, and 168 hpi. Control BR/08 virus-infected immunosuppressed BALB/c mice were given a single dose of heterologous anti-H6 MAb (8H9) that was i.p. administered. The probabilities of survival were determined by Kaplan-Meier and log-rank tests.

TABLE 1.

Effect of MAb monotherapy on antibody responses in immunosuppressed BALB/c mice after primary and secondary challenge with BR/08 influenza virus

Treatment regimena			No. survived/total no. (%)	Mean survival day ± SD	Antibody response after primary BR/08 virus challenge (GMT ± SD)b			Antibody response after secondary BR/08 virus challenge (GMT ± SD)b
Group	Dose (mg/kg)	No. of admin.			HI titer	VN titer	NI titer	HI titer	VN titer	NI titer
Monotherapy with anti-HA (1D2) MAb	1	1	0/8 (0)	11.3 ± 3.8	N/Ac	N/A	N/A	N/A	N/A	N/A
	5	1	5/8 (62.5)	22.3 ± 9.3	<d	6.3 ± 0.0	<e	8.3 ± 1.0	12.3 ± 0.0	11.6 ± 0.0
	25	1	7/8 (87.5)	27.3 ± 4.9	<	6.5 ± 0.4	<	7.0 ± 0.5	12.3 ± 0.0	11.3 ± 0.8
	5	2	6/8 (75.0)	25.3 ± 7.0	<	6.5 ± 0.4	<	7.0 ± 0.5	12.3 ± 0.0	11.6 ± 1.0
	5	3	7/8 (87.5)	26.5 ± 7.1	<	6.5 ± 0.4	<	7.4 ± 0.7	12.3 ± 0.0	11.6 ± 0.0
Monotherapy with anti-NA (1F2) MAb	1	1	7/8 (87.5)	27.0 ± 5.7	<	7.2 ± 0.0	5.6f	6.9 ± 0.5	12.3 ± 0.0	9.3 ± 2.3
	5	1	8/8 (100)	29.0 ± 0	<	7.7 ± 0.5	6.6f	7.3 ± 0.9	12.3 ± 0.0	9.2 ± 2.0
	25	1	8/8 (100)	29.0 ± 0	<	8.1 ± 0.5	8.5 ± 1.0	7.8 ± 0.5	12.3 ± 0.0	8.5 ± 0.9
	5	2	8/8 (100)	29.0 ± 0	<	8.2 ± 0.4	7.0 ± 0.5	7.4 ± 0.8	12.3 ± 0.0	8.0 ± 1.8
	5	3	7/8 (87.5)	26.3 ± 7.8	<	8.2 ± 0.4	8.4 ± 0.7	7.9 ± 0.5	12.3 ± 0.0	9.2 ± 1.8
Control treatment
8H9	25	1	0/8 (0)	9.1 ± 1.8	N/A	N/A	N/A	N/A	N/A	N/A
Peramivir	75	3	0/8 (0)	11.0 ± 1.3	N/A	N/A	N/A	N/A	N/A	N/A

^aGroups (n = 8/group) of immunosuppressed BALB/c mice were intranasally inoculated with 10 MLD₅₀ of BR/08 influenza virus. Monotherapy with the anti-HA MAb 1D2 or the anti-NA MAb 1F2 was i.p. administered as a single dose (1, 5, or 25 mg/kg) or repeated doses (5 mg/kg) at 24 hpi. Control BR/08 virus-infected immunosuppressed BALB/c mice were given either a single dose (25 mg/kg) of heterologous anti-H6 (8H9) MAb i.p. administered or repeated doses (75 mg/kg) of NAI peramivir administered by i.m. injection. admin., administrations.

^bHemagglutination inhibition (HI), virus-neutralizing (VN), and NA inhibition (NI) titers were determined for mouse sera collected 27 dpi after primary challenge with 10 MLD₅₀ of BR/08 influenza virus (2 days before secondary infection) and 20 dpi after secondary challenge with 10 MLD₅₀ of BR/08 influenza virus. HI, VN, and NI titers are expressed as the geometric mean titer (GMT) under log₂ of the reciprocal titer ± SD.

^cN/A, not applicable (all mice in the group died).

^d<, less than 1:20 (HI titer).

^e<, less than 1:50 (NI titer).

^fOnly one of the surviving animals had NI titers.

Next, we assessed the efficacy of combination treatment with 1D2 and 1F2 in immunosuppressed BALB/c mice when the treatment window was extended and initiated at 24 hpi or delayed until 48 or 72 hpi (Fig. 1B, D, and E). When initiated at 24 hpi, treatment of mice with combined 1D2 (10 mg/kg) and 1F2 (1 mg/kg) MAbs resulted in 86% survival, with a mean survival of 24.1 days (Table 2). This survival rate was similar to that obtained with 1F2 monotherapy (87.5%) but was higher than that obtained with 1D2 monotherapy (75%). When initiated at 48 hpi, treatment of mice with combined 1D2 and 1F2 resulted in 87.5% survival, with a mean survival of 24.5 days (Table 2); this survival rate indicated that the protection conferred by combination therapy was greater than that resulting from monotherapy with either of the MAbs. When treatment was initiated at 72 hpi, both combination therapy and monotherapy provided 50% protection (Fig. 1E). Thus, combination treatment with 1D2 and 1F2 provided greater protection than monotherapy when started 24 to 48 h after influenza B virus inoculation.

TABLE 2.

Effect of MAb monotherapy and combination treatment on antibody responses in immunosuppressed BALB/c mice after primary challenge with BR/08 influenza virus

Treatment regimena			No. survived/total no. (%)	Mean survival day ± SD	Antibody response after primary BR/08 virus challenge (GMT ± SD)b
Group	Dose (mg/kg)	No. of admin.			HI titer	VN titer	NI titer
Initiation of MAb treatment (24 hpi)
1D2	10	1	6/8 (75.0)	22.1 ± 9.1	<c	5.8 ± 0.5	<d
1F2	1	1	7/8 (87.5)	25.3 ± 4.9	<	6.7 ± 0.5	<
1D2 + 1F2	10 + 1	1	6/7 (86.0)	24.1 ± 7.6	<	6.6 ± 0.5	<
Initiation of MAb treatment (48 hpi)
1D2	10	1	5/8 (63.0)	19.9 ± 9.9	<	5.9 ± 0.5	<
1F2	1	1	4/8 (50.0)	17.8 ± 10.1	<	6.8 ± 0.6	<
1D2 + 1F2	10 + 1	1	7/8 (87.5)	24.5 ± 7.1	<	6.9 ± 0.5	<
Initiation of MAb treatment (72 hpi)
1D2	10	1	4/8 (50.0)	17.4 ± 10.3	<	5.8 ± 0.6	<
1F2	1	1	4/8 (50.0)	17.8 ± 10.0	<	6.1 ± 0.5	<
1D2 + 1F2	10 + 1	1	4/8 (50.0)	18.3 ± 9.6	<	6.8 ± 0.6	<
Control treatment
8H9	5	1	0/8 (0)	8.3 ± 0.9	N/Ae	N/A

^aGroups (n = 7–8/group) of immunosuppressed BALB/c mice were intranasally inoculated with 10 MLD₅₀ of BR/08 influenza virus. Mice received monotherapy with the anti-HA MAb 1D2 or the anti-NA MAb 1F2 i.p. administered as a single dose (10 and 1 mg/kg, respectively) or they received combination therapy with both MAbs at 24, 48, or 72 hpi. Control BR/08 virus-infected immunosuppressed BALB/c mice were given a single dose (5 mg/kg) of heterologous anti-H6 (8H9) MAb i.p. administered.

^bHemagglutination inhibition (HI), virus-neutralizing (VN), and NA inhibition (NI) titers were determined for mouse sera collected 27 dpi after primary challenge with 10 MLD₅₀ of BR/08 influenza virus. HI, VN, and NI titers are expressed as the geometric mean titer (GMT) under log₂ of the reciprocal titer ± SD.

^c<, less than 1:20 (HI titer).

^d<, less than 1:50 (NI titer).

^eN/A, not applicable (all mice in the group died).

Influenza BR/08 virus replication in the tissues of MAb-treated immunosuppressed mice and selection of escape variants.

To examine whether the MAbs 1D2 and 1F2, as monotherapy or combination therapy, could control influenza virus replication in immunosuppressed mice, lungs of infected animals were collected at 3, 9, 16, and 30 dpi (Fig. 2), and nasal turbinates were collected at 9 dpi (see Fig. S3 in the supplemental material). Because of the early death of the control animals (virus-inoculated, anti-H6 HA [8H9] MAb-treated mice), BR/08 virus replication in these groups was determined only at 3 and 9 dpi, and the titers were between 4.5 and 5.0 log₁₀ 50% tissue culture infectious dose per milliliter (TCID₅₀/ml), respectively (Fig. 2A and B). Treatment with 1D2 and 1F2 at all doses tested (1, 5, and 25 mg/kg) tended to decrease BR/08 virus replication in the lungs of immunosuppressed mice, compared with that of control animals, at 9 dpi. However, these differences in viral titers were not statistically significant. Treatment with the anti-HA MAb 1D2 did not significantly decrease virus titers at 16 dpi (average titer, 4 log₁₀ TCID₅₀/ml) but did contribute to the restriction of virus replication at 30 dpi (Fig. 2A). Consistent with the survival benefits of anti-NA MAb 1F2, virus titers in the lungs of treated animals were below the detection limit at 16 and 30 dpi, suggesting that virus clearance is enhanced with this regimen, compared with the 1D2 regimen. The only exception was observed with the lowest dose (1 mg/kg) of 1F2, which was unable to clear the virus from the lungs of mice at 16 dpi. When treatment was initiated at 24, 48, or 72 hpi, the BR/08 virus titers in the lungs of mice treated with 1D2 and 1F2 in combination did not differ from those in the lungs of mice in the control and monotherapy groups on 9 dpi, suggesting that there was no benefit with respect to reducing virus replication in combination therapy (Fig. 2C). Viral replication in the nasal turbinates of MAb-treated mice corresponded with that in the lungs, except that slightly higher tissue titers were obtained in the nasal region (Fig. S3).

FIG 2.

Influenza BR/08 virus load in the lungs of immunosuppressed mice treated with MAbs. Immunosuppressed BALB/c mice were inoculated and treated as described in the legend for Fig. 1. Virus titers were determined in the lungs of mice (n = 3/group), beginning at 3, 9, 16, and 30 dpi, by TCID₅₀ assays in MDCK cells. Mice received monotherapy with the anti-HA MAb 1D2 (A) or the anti-NA MAb 1F2 (B) or were treated with a combination of the two (C). The bars represent the mean virus titer (expressed as the log₁₀TCID₅₀/ml ± SD). Dashed lines indicate the minimum level of virus detection (1 log₁₀TCID₅₀/ml). *, P < 0.05, comparing treatment and control groups. Probabilities were determined by one-way ANOVA with Bonferroni’s multiple-comparison posttest.

A possible selection of BR/08 virus escape variants under MAb treatment of immunosuppressed mice was assessed by variant analysis of next-generation sequencing (NGS) data obtained from the nasal turbinates of mice that received both monotherapy and combination therapy. This analysis revealed no substitutions in the HA and NA surface glycoproteins associated with the emergence of escape variants (see Tables S1 and S2 in the supplemental material).

Histomorphometry of the lungs of immunosuppressed mice lethally challenged with influenza BR/08 virus and treated with MAbs.

We observed no significant differences in virus titers in MAb-treated animals compared with those of controls, but the MAb-treated animals ultimately had favorable survival outcomes. To account for this discrepancy, we hypothesized that the MAbs decreased the severity and extent of pulmonary BR/08 virus infection. Therefore, whole-lung sections obtained from control and MAb-treated groups at 9 dpi were examined histologically (Fig. 3). The lungs of BR/08-infected control animals exhibited prominent perivascular and intra-alveolar inflammation with extensive distribution of viral antigens, indicating a widespread active infection (involving 68% of the lung area) (Fig. 3B). Peramivir treatment alone did not markedly alter the severity or extent of virus infection (Fig. 3C and D). When treatment was initiated at 24, 48, or 72 hpi, 1D2 reduced the extent of infection in mouse lungs to 28%, 26%, and 27%, respectively (Fig. 3F, L, and R). Treatment with 1F2 was more effective than treatment with 1D2 at controlling BR/08 virus spread, with the most pronounced effect being observed when therapy began at 24 hpi (15%) (Fig. 3H). Treatment-associated reduction of virus spread in the lungs was slightly less effective at 48 and 72 hpi (20% and 27%, respectively). Immunotherapy with a combination of 1D2 and 1F2 markedly reduced the number of intact virus-positive cells and the amount of virus antigen detected in the lungs to 6%, 10%, and 10% when treatment was initiated at 24, 48, and 72 hpi, respectively (Fig. 3J, P, and V). The combination of anti-HA and anti-NA MAbs was, therefore, the most effective regimen for reducing damage and the spread of influenza BR/08 virus in the lungs of immunocompromised mice.

FIG 3.

Pulmonary histopathologic changes in influenza BR/08 virus-infected immunosuppressed mice treated with MAbs. Immunosuppressed BALB/c mice (n = 5/group) were intranasally inoculated with 10 MLD₅₀ of influenza BR/08 virus. The control mice received either a single dose (5 mg/kg) of heterologous anti-H6 MAb (8H9) i.p. administered at 24 hpi (A, B) or three doses (75 mg/kg) of NAI peramivir administered by i.m. injection at 24, 96, and 168 hpi (C, D). Mice were treated with the anti-HA MAb 1D2 (E, F, G, H, I, J) or the anti-NA MAb 1F2 (K, L, M, N, O, P) or a combination of the two (Q, R, S, T, U, V). Monotherapy with 1D2 or 1F2 and combination treatment were i.p. administered as a single dose (10 and 1 mg/kg, respectively) beginning at 24, 48, or 72 hpi. Mouse lungs were fixed in 10% neutral-buffered formalin, subjected to immunohistochemical (IHC) staining with anti-HA influenza B virus antiserum, and analyzed by histomorphometry. Magnification, ×20 (IHC) or ×2 (histomorphometry). Histomorphometry images stained for viral antigen were scanned using a ScanScope XT slide scanner (Aperio, Technologies, Vista, CA). The total lung areas examined are outlined in green, and areas of active infection with antigen-positive cells are shown in red. The images are representatives of examined animals and show the histopathologic changes and the extent of BR/08 virus infection at 9 dpi. The percentage of the total lung area containing viral antigen is indicated for each image.

Production of pulmonary cytokines and chemokines.

The pulmonary cytokines and chemokines are critical markers of innate immune responses and are activators of adaptive immune responses that can define the outcome during lethal viral infections (25, 26). We assessed the effect of MAb treatment on the expression of 25 cytokines and chemokines in the lungs of immunosuppressed mice at 9 dpi. Because of the high variability of the expression profiles, we divided a heat-map into low (2- to 10-fold) and high (11- to 55-fold) fold changes (Fig. 4A and B, respectively). The time of treatment initiation after virus infection appears to affect the expression of cytokines and chemokines. When treatment was initiated at 24 hpi, 1D2 increased the expression of IL-12P40 and 1F2 increased the expression of interleukin-9 (IL-9), IL-12P70, IP-10, KC, and MCP-1 (Fig. 4). The pulmonary concentrations of IP-10 and MCP-1 were increased 11.1-fold and 22.5-fold, respectively, by 1F2 treatment but not by the combination treatment when it was initiated at 24 hpi (see Fig. S4 in the supplemental material). When treatment was initiated at 48 hpi, the greatest effect of 1D2 and 1F2 as a monotherapy or combination therapy was on the expression of IP-10 (with increases of 18.7-, 22.6-fold for monotherapy, and 40-fold for combination, respectively) and MCP-1 (with increases of 16-, 28.1-fold for monotherapy, and 38.9-fold for combination, respectively) (Fig. 4B; Fig. S4). Additionally, combination treatment induced a 10.7-fold increase in the expression of KC. When treatment was initiated at 72 hpi, 1D2 had the greatest effect on the expression of RANTES (with a 6.8-fold increase). RANTES is a potent chemoattractant that plays a critical role in the immune response to viral infections. The high expression of RANTES was diminished when 1D2 was administered with 1F2 (see Fig. S5 in the supplemental material). Treatment with 1D2 and 1F2 resulted in 10- and 20-fold increases in the concentrations of proinflammatory IP-10 and MCP-1, whereas the combination treatment increased their concentrations 24.4- and 52.8-fold, respectively (Fig. 4B). The fold change in the expression of all the other cytokines and chemokines remained between 0.2- and 3.9-fold (Fig. 4A). An increased expression of IP-10 and MCP-1 in groups receiving combination treatment, compared with monotherapy, can potentially promote a better control of virus spread within the lungs.

FIG 4.

Pattern of pulmonary cytokine and chemokine expression in influenza BR/08 virus-infected immunosuppressed mice treated with MAbs. Twenty-five cytokines and chemokines were assayed in lung homogenates (n = 3/group) at 9 dpi by using a Myctomag-70K-PMX Milliplex MAP mouse cytokine/chemokine panel (Millipore). The expression of analytes is normalized to that in the control group (anti-H6 MAb, 8H9). Results for all the 1D2 and 1F2 monotherapy and combination treatment groups were plotted in the heatmap as the fold change compared with that of the control group. The heatmap is monochromatic; the intensity of the blue color is directly proportional to the fold change increase in the expression of the analyte. Analytes with fold changes of 1 to 10 (A) and with fold changes of 11 to 55 (B) are shown.

Seroconversion after primary and secondary challenges with BR/08 virus.

Lymphocyte and peripheral neutrophil counts in immunosuppressed mice are markedly decreased due to dexamethasone (DEX) and cyclophosphamide (CP) treatment (see Fig. S6 in the supplemental material). However, by day 20 after immunosuppression, immune cell counts started to recover and were almost back to normal at day 35 posttreatment. To examine the development of humoral immune responses in MAb-treated immunosuppressed mice after primary BR/08 infection, we determined the hemagglutination inhibition (HI), NA inhibition (NI), and virus neutralization (VN) titers against homologous virus in mouse sera collected at 30 dpi. In mice treated with 1D2, both HI and NI active antibodies were below the level of detection, but VN titers were determined to be in the range of 6.3 to 6.5 (Table 1). In mice treated with 1F2, no HI active antibodies were detected, but NI titers were obtained ranging from 5.6 to 8.5; only one of the surviving mice which received 1 or 5 mg/kg 1F2 had a detectable NI titer (Table 1). VN titers (range, 7.2 to 8.2) were also detected, which were 1 to 2 log higher than those obtained from 1D2-treated mice groups. Mice treated with 1D2 and 1F2 in combination did not develop anti-HA or anti-NA antibodies, even when treatment initiation was delayed by 48 or 72 hpi; however, VN titers were detected and remained unchanged between any two delayed-treatment time points (range, 6.6 to 6.9) (Table 2). As with results obtained from monotherapies (Table 1), VN titers from 1F2 and the 1D2 and 1F2 combination were substantially higher than those obtained from 1D2 alone.

To study the level of protection that could be obtained after MAb-treated mice recovered from primary BR/08 virus infection, we rechallenged mice with BR/08 virus at 29 dpi. No deaths within 14 days of observation were observed in any MAb-treated mice that received a secondary challenge with BR/08 (data not shown). Serum titers to homologous virus were comparable between the 1D2- and 1F2-treatment groups and were 6.9 to 8.3 for HI, 8 to 11.6 for NI, and 12.3 for VN at 27 days after secondary challenge with BR/08 (Table 1). Taken together with the recovery of lymphocytes and neutrophils around this time point (Fig. S6), these results indicate that the presence of VN antibodies accompanied by the rebound of immune cells were instrumental in protecting mice from secondary challenge with BR/08.

DISCUSSION

This study is the first to investigate the efficacy of MAbs in monotherapy and combination treatment of influenza B virus infection in immunosuppressed mice. It was conducted according to the hypothesis that administering anti-influenza MAbs to an immunosuppressed host would augment endogenous immune responses, ameliorate clinical illness, and protect the host from influenza virus-induced mortality. We have demonstrated that treatment with MAbs targeting the HA and NA proteins substantially protected immunosuppressed mice from lethal challenge with influenza B virus. A combined treatment with anti-HA and anti-NA MAbs, compared with monotherapy regimens, reduced the severity of the infection and increased the survival of the immunosuppressed mice. An emergence of escape variants over the course of MAb treatments of mice was not observed.

In high-risk patient groups, viral infections like influenza B can be quite severe or life-threatening, and the consideration of treatment options must be broadened to provide the best possible outcomes. Antiviral drug options for treating influenza B virus infection in immunocompetent patients are limited to NAIs and the PA inhibitor BXM, although there have been reports that both drugs have reduced effectiveness against influenza B virus infection, compared with influenza A virus infection (11–13). The available preclinical data indicate that the NAIs peramivir and oseltamivir, the standard-of-care for influenza, were ineffective at protecting genetically modified immunocompromised and pharmacologically immunosuppressed mice from influenza B virus infection (27, 28), thus demonstrating that NAIs have limited efficacy in immunocompromised mouse models. With respect to BXM, the baseline 50% effective concentration is higher for influenza B viruses than that for influenza A viruses (29, 30). Such differences in antiviral activity have been also shown in vitro and in immunocompetent BALB/c mice (31). However, there is no data regarding the anti-influenza B virus activity of BXM in the context of immunocompromised animals.

One alternative is immunotherapy with MAb- and polyclonal antibody-based intravenous immunoglobulin (hIVIG) (32). The rationale for choosing immunotherapy is based on the known course of natural influenza virus infection, where the development of humoral immune responses appears to correlate temporally with clinical recovery. Unlike antiviral drug therapy in which the drug often requires conversion to a metabolic form and has a distinct target for virus inhibition, the potential mechanisms by which MAbs confer protection can be diverse and include direct antiviral action, promoting cytokine/chemokine expression, and recruiting immune cells. To date, using MAb and hIVIG therapy in immunocompetent patients has been met with mixed results, particularly for influenza A virus therapy (33–36). In a phase 2, placebo-controlled study conducted in a double-blind manner in hospitalized patients with severe influenza A virus infection, treatment with the human MAb MHAA4549A (targeting the HA stalk region of influenza A virus) resulted in a significant reduction in clinical symptoms and viral burden compared with that seen in patients treated with placebo (33). However, two other clinical studies demonstrated that treatment with MHAA4549A did not significantly reduce the time to clearance of the viral load and did not demonstrate a clinical benefit compared with NAI oseltamivir alone in hospitalized patients with severe influenza A virus infection (34). The MAb VIS410 (which also targets the HA stalk region of influenza A viruses) proved safe and efficacious in a phase 2a influenza A(H1N1) viral challenge study (35). VIS410 was safe and well tolerated in adults with uncomplicated influenza A infection, having favorable effects on symptom resolution and virus replication (36). Fortunately, current data suggest that hIVIG has a greater efficacy against influenza B infection than against influenza A infection (32).

MAbs with proven neutralization activity, such as the 1F2 MAb used in this study, can block the activity of the NA enzyme and, thus, regulate virus replication and spread. Other MAbs that do not possess neutralization activity, such as the 1D2 MAb used in this study, can protect through Fc-mediated effector functions (22). One strategy to improve upon MAb therapy is to use a cocktail of antibodies targeting multiple proteins and/or antigenic sites. Here, we used antibodies targeting two major surface proteins (HA and NA), potentially blocking different stages of the influenza virus replication cycle. Although the mechanism of action of anti-HA nonneutralizing MAbs is not completely understood (22), enhanced influenza virus clearance could be one mechanism of protection by anti-NA antibodies, as 1F2 consistently induced improved VN titers in immunosuppressed mice. Our results show that a two-dose 5-mg/kg regimen did not improve survival rate. Therefore, the optimal dosing for 1F2 monotherapy in immunosuppressed mice needs to be further examined. The current influenza virus vaccines do not contain an NA component (as is the case with the Flublok vaccine), contain a very small amount of a NA component, or do not efficiently present important conserved and protective NA epitopes (37). Therefore, the coadministration of anti-NA MAbs may have synergistic protective effects that are superior to those obtained with anti-HA monotherapy alone.

The expression of proinflammatory cytokines/chemokines can be instrumental in controlling influenza virus infection (38); in the immunosuppressed host, even high concentrations of cytokines/chemokines may not result in a cytokine storm but instead lead to better control of infection. The increased concentrations of IP-10 and MCP-1 chemokines observed during MAb combination therapy could limit the immunopathologic lung lesions associated with influenza infection in an immunosuppressed host. IP-10/CXCL10 attracts immune cells to the lung and are important for T-cell activation (39). Similar activity has been reported for MCP-1 (CCL2) (40). Recruitment of immune cells to the site of infection is a powerful mechanism for protection. However, the transient lymphopenia and neutropenia due to DEX and CP treatment suggest that other immune cells (e.g., natural killer [NK] cells) could be responsible for the cytokine/chemokine response. Infection of the BR/08 virus is associated with extensive pulmonary immunopathology and extended induction of proinflammatory cytokine/chemokine rather than aberrant virus replication (27, 28). The lack of antibodies elicited by influenza B virus infection in these immunosuppressed mice was complemented by the MAb treatment and contributed in modulating the deleterious BR/08-associated immunopathology and cytokine/chemokine responses, which are more efficiently facilitated by the 1D2 and 1F2 combination regimen. Moreover, the development of virus-neutralizing antibodies and recovery of immune cell counts ameliorated protection of mice from secondary challenge with influenza B virus.

A perplexing observation in our study is the higher retention levels of administered MAbs at day 1 postadministration and the overall shorter half-life of the MAbs in the immunosuppressed mice than those in the controls. The explanation for this is unknown, but it is possible that defects in the immune system affect cellular Fc expression, antibody distribution through the interstitial space, and antibody recycling through the neonatal Fc receptor (41, 42), leading to a reduction in MAb bioavailability and/or clearance capacity. However, MAbs can be engineered to extend their half-life and to improve their therapeutic efficacy (43, 44), and it is possible these techniques could be applied to 1D2 and 1F2 once the mechanism underlying these observations is fully understood.

Ultimately, more preclinical studies are required in order to understand the role of MAbs and to improve antibody therapy for influenza virus infections. In particular, the use of these approaches in immunocompromised animals and with influenza B viruses has been a neglected area of study. Our preclinical data suggest that there is a role for MAbs and especially for combination therapy with antibodies that target both the HA and the NA in the treatment of influenza in immunocompromised individuals.

MATERIALS AND METHODS

Viruses, cells, and compounds.

Influenza BR/08 virus (B/Victoria/2/1987-like lineage) was obtained from the Influenza Division at the Centers for Disease Control and Prevention and was grown in 10-day-old embryonated chicken eggs for 72 h at 33°C. The 50% tissue culture infectious dose per milliliter (TCID₅₀/ml) was determined in Madin-Darby canine kidney (MDCK) cells (American Type Culture Collection, Manassas, VA) in the presence of tosyl phenylalanyl chloromethyl ketone (TPCK)-treated trypsin. The NAI peramivir [(1S,2R,3R,4R)-3-(1-acetamido-2-ethyl-butyl)-4-(diaminomethylideneamino)-2-hydroxy-cyclopentane-1-carboxylic acid] was dissolved in sterile phosphate-buffered saline (PBS) and filter sterilized, and stocks were stored at −20°C until use. Dexamethasone (DEX) and cyclophosphamide (CP; Sigma-Aldrich, St. Louis, MO) were dissolved in sterile PBS.

Preparation of MAbs.

The MAbs (1D2, isotype IgG2b; 3F4, 4C2, 1F2, all of isotype IgG2a) were prepared as described previously (22, 23). Briefly, female 6-week-old BALB/c mice (The Jackson Laboratory, Bar Harbor, ME) were anesthetized with isoflurane and inoculated intranasally with 10⁵ TCID₅₀/mouse of B/Lee/1940, B/Victoria/2/1987 (Victoria lineage), and B/Yamagata/16/1988 (Yamagata lineage) viruses in a 50-μl volume. Broadly cross-reactive nonneutralizing anti-HA and anti-NA MAbs were selected by enzyme-linked immunosorbent assay (ELISA). A control heterologous anti-H6 MAb (8H9, isotype IgG2a) was generated in a similar manner (22, 23). The antibodies were purified as described earlier (22). Briefly, hybridoma clones producing each of these antibodies were grown in Hybridoma-SFM serum-free medium and scaled up to a volume of 500 ml. After low-speed centrifugation (4,000 × g at 4°C for 10 min) and sterile filtration (with a 0.22-μm filter), the antibodies were purified using a column packed with Protein G Sepharose 4 fast flow beads (GE Healthcare). Elution was carried out with 0.1 M glycine (pH 2.7), and the eluate was neutralized with 2 M Tris-HCl (pH 9.4), after which buffer exchange to PBS at pH 7.4 was carried out. Purified antibodies were quantified using a NanoDrop device (Thermo Scientific) by measuring the optical density at 280 nm. The monoclonal antibody 1D2 (KL-BHA-1D2) is a broadly cross-reactive antibody targeting the influenza B virus HA head, whereas 1F2 (KL-BNA-1D2) is a broadly cross-reactive antibody targeting the influenza B virus NA.

MAb treatment of immunosuppressed mice.

Female 6-week-old BALB/c mice (The Jackson Laboratory, Bar Harbor, ME) were treated with DEX and CP to induce immunosuppression (27). Ten doses of DEX (10 mg/kg in 0.2 ml/mouse) were intraperitoneally (i.p.) administered every day starting 24 h before virus inoculation. Two doses of CP (150 mg/kg in 0.3 ml/mouse) were i.p. administered at −24 and 120 hpi. Differential leukocytes were enumerated and analyzed in whole blood with a Forcyte hematology analyzer (Oxford Science, Oxford, CT). Immunosuppressed mice were anesthetized with isoflurane and inoculated intranasally with 10 50% mouse lethal doses (MLD₅₀) of BR/08 influenza virus in 30 μl of PBS. In a pilot study, treatment of immunocompromised BALB/c mice (n = 8/group) was initiated at 24 hpi with three anti-HA MAbs (1D2, 3F4, and 4C2) or an anti-NA MAb (1F2) i.p. administered as a single dose (at 24 hpi) or a double dose (at 24 and 192 hpi) of 5 mg/kg. The anti-HA MAb 1D2 and anti-NA MAb 1F2 were selected for further studies. Monotherapy of immunosuppressed BALB/c mice (n = 8/group) with 1D2 or 1F2 was administered at 24 hpi as a single dose (1, 5, or 25 mg/kg in 0.1 ml/mouse). For repeated-dose regimens, 1D2 and 1F2 were administered to mice (n = 8/group) at a 5-mg/kg dose either twice (at 24 and 72 hpi) or three times (at 24, 96, and 168 hpi) after BR/08 virus inoculation. The control mice (n = 8) received either a single dose (25 mg/kg in 0.1 ml/mouse) of a heterologous MAb against H6 HA of influenza A virus (MAb 8H9) i.p. administered at 24 hpi or three doses (75 mg/kg in 0.1 ml/mouse) of the NAI peramivir administered by intramuscular (i.m.) injection at 24, 96, and 168 hpi. In the combination treatment experiments, 1D2 (10 mg/kg) was coadministered with 1F2 (1 mg/kg) to mice (n = 8/group) at 24, 48, or 72 hpi, and the results were compared with those obtained with MAb monotherapy given on the same schedule. The survival of each mouse was monitored daily for 30 dpi, and any animal that showed signs of severe disease and/or a 30% weight loss was euthanized.

Reinfection.

All immunosuppressed BALB/c mice that survived lethal infection with influenza BR/08 virus while being treated with the MAbs 1D2 and 1F2 were anesthetized with isoflurane and received a secondary challenge with 10 MLD₅₀/mouse of influenza BR/08 virus at 35 dpi. Age-matched naive female BALB/c mice (n = 5/group) were used as controls.

Virus lung and nasal turbinate titers.

To determine the level of virus replication, additional mice (n = 3/group/time point) from each experimental group were euthanized at 3, 9, 16, and 30 dpi (for monotherapy groups) or at 9 dpi (for monotherapy and combination therapy groups). Their nasal turbinates (9 dpi only) and lungs were removed, thoroughly rinsed with sterile PBS, and homogenized, and the homogenate was suspended in 1 ml of cold PBS. The suspensions were cleared by centrifugation at 2,000 × g for 10 min, and then the virus titers were determined in MDCK cells and expressed as the mean log₁₀TCID₅₀/ml ± the standard deviation (SD).

Cytokine and chemokine analysis.

At 3, 9, 16, and 30 dpi, the concentrations of each of 25 cytokines and chemokines were measured in lung homogenates (n = 3/group) by using a Myctomag-70K-PMX Milliplex MAP mouse cytokine/chemokine panel (Millipore) in accordance with the manufacturer’s instructions. The plates were read on a Luminex 100/200 analyzer using the xPonent software. The expression of the 25 cytokines and chemokines was normalized to that in the control group (anti-H6 HA MAb, 8H9).

Lung histopathology and immunohistochemistry.

Lungs were collected at 9 dpi (n = 5/group) and infused with 10% neutral-buffered formalin (Thermo Scientific), and then tissue sections were either stained with hematoxylin and eosin or subjected to immunohistochemical staining after undergoing antigen retrieval for 30 min at 98°C. An anti-HA goat antiserum (against B/Florida/04/2006, B/Yamagata/16/1988-like–lineage virus) diluted 1:2,000 was used as the primary antibody, and a biotinylated donkey anti-goat antibody (catalog no. sc-2042; Santa Cruz Biotechnology, Santa Cruz, CA) diluted 1:200 was used as the secondary antibody. The Discovery ChromoMap DAB kit (Ventana Medical Systems) was used as the chromogenic substrate, and stained sections were examined by a pathologist who was blind to the experimental group assignments. The extent of virus spread in the lungs was quantified by first capturing digital images of whole-lung sections stained for viral antigen by using a ScanScope XT slide scanner (Aperio Technologies, Vista, CA) and then manually outlining alveolar parenchyma containing virus antigen-positive pneumocytes. The extent of virus infection was quantified as a percentage of the total alveolar lung field by using ImageScope software (Aperio Technologies) (27).

Determination of antibody half-life.

Female 6-week-old immunocompetent and immunosuppressed BALB/c mice (n = 5/group) were given a single i.p. dose (5 mg/kg) of 1D2 or 1F2 MAb. Blood was collected by retro-orbital bleeding into serum separation tubes (Becton, Dickinson, Franklin Lakes, NJ) at 24 h after MAb administration and every 7 days thereafter until day 50 after treatment. The serum samples were stored at −20°C until use. The half-life was determined by enzyme-linked immunosorbent assay (ELISA) with recombinant HA and NA proteins (0.1 μg/100 μl of serum, produced in the baculovirus system [45]) derived from the B/Malaysia/2506/2004 and B/Yamagata/16/1988 viruses, respectively. The endpoint titers were determined, and the half-life was calculated using GraphPad Prism 8.0 software.

Next-generation sequencing.

Viral RNA was extracted from nasal turbinate homogenates collected at 9 dpi using the QIAamp viral RNA minikit (Qiagen) according to the manufacturer’s instructions. Nasal turbinates were chosen as the sample types on which to perform NGS of the HA and NA genes due to the higher virus titers needed to attain a minimum depth of 1,000 reads through the entire length of each gene and variant calling above 5% frequency. The HA and NA genes were amplified using the one-step SuperScript III reverse transcriptase PCR (RT-PCR) kit (Invitrogen) and segment-specific primers (46). NGS of purified products was conducted, and libraries were prepared using a Nextera XT DNA library preparation kit (Illumina) at the recommended volumes. The libraries were then run on the MiSeq platform (Illumina), the sequences were analyzed by CLC Genomics Workbench 12, and a comparison was done against the BR/08 HA and NA sequences (Global Initiative on Sharing All Influenza Data accession numbers EPI172555 [HA] and EPI172554 [NA]).

Serologic assays.

Sera were collected by retro-orbital bleeding at 27 dpi, treated with a receptor-destroying enzyme (Denko-Seiken, San Jose, CA), heat-inactivated at 56°C for 1 h, and tested by hemagglutination inhibition (HI) assay with 0.5% turkey red blood cells (Rockland Immunochemicals, Pottstown, PA). Enzyme-linked lectin assays (ELLAs), used to determine the NA inhibition (NI) activity of mouse sera, were performed as described previously (47–49). Virus-neutralizing (VN) antibody titers in mouse sera were determined in MDCK cells as described previously (50).

Statistical analysis.

Virus titers, cell counts, and cytokine/chemokine levels were compared by one-way analysis of variance (ANOVA) with Bonferroni’s posttest (GraphPad Prism 8.0 software). The Kaplan-Meier method was used to estimate the probability of survival, and the log-rank test was used to compare survival rates.

Ethics statement.

All animal experiments were performed in an animal biosafety level 2+ (ABSL2+) containment facility approved by the U.S. Department of Agriculture. All studies were conducted in accordance with the policies of the National Institutes of Health and the Animal Welfare Act and after approval by the Institutional Animal Care and Use Committee.

Data availability.

Data on variant analysis of NGS data obtained from the nasal turbinates of mice and cytokine/chemokine expression profiles of mice that received both monotherapy and combination therapy are stored at St. Jude Children’s Research Hospital and will be available upon request.