Mechanism of Action

Palivizumab, a recombinant humanized monoclonal antibody which provides passive immunity against RSV, acts by binding the RSV envelope fusion protein (RSV F) on the surface of the virus and blocking a critical step in the membrane fusion process. Palivizumab also prevents cell-to-cell fusion of RSV-infected cells. 

Antiviral Activity

The antiviral activity of palivizumab was assessed in a microneutralization assay in which increasing concentrations of antibody were incubated with RSV prior to addition of the human epithelial cells HEp-2. After incubation for 4-5 days, RSV antigen was measured in an ELISA assay. The neutralization titer (50% effective concentration [EC50]) is expressed as the antibody concentration required to reduce detection of RSV antigen by 50% compared with untreated virus-infected cells. Palivizumab exhibited median EC50 values of 0.65 mcg per mL (mean 0.75 ± 0.53 mcg per mL; n=69, range 0.07-2.89 mcg per mL) and 0.28 mcg per mL (mean 0.35 ± 0.23 mcg per mL; n=35, range 0.03-0.88 mcg per mL) against clinical RSV A and RSV B isolates, respectively. The majority of clinical RSV isolates tested (n=96) were collected from subjects across the United States (CA, CO, CT, IL, MA, NC, NY, PA, RI, TN, TX, VA), with the remainder from Japan (n=1), Australia (n=5) and Israel (n=2). These isolates encoded the most common RSV F sequence polymorphisms found among clinical isolates worldwide. 

Palivizumab serum concentrations of greater than or equal to 40 mcg per mL have been shown to reduce pulmonary RSV replication in the cotton rat model of RSV infection by 100-fold. 

Resistance

Palivizumab binds a highly conserved region on the extracellular domain of mature RSV F, referred to as antigenic site II or site A, which encompasses amino acids 262 to 275. All RSV mutants that exhibit resistance to palivizumab have been shown to contain amino acid changes in this region on the F protein. 

F protein sequence variations within antigenic site A: Amino acid substitutions in antigenic site A selected either in cell culture, in animal models, or in human subjects that resulted in palivizumab resistance were N262D, N268I, K272E/M/N/Q/T, and S275F/L. RSV variants expressing the K272N substitution in F protein showed a 5164 ± 1731-fold decrease in susceptibility (i.e., fold increase in EC50 value) when compared to the wild-type RSV, while variants containing the N262D, S275F/L, or K272E/M/Q/T substitutions showed a greater than 25,000-fold decrease in susceptibility to palivizumab. The N268I substitution conferred partial resistance to palivizumab; however, fold changes in susceptibility were not quantified for this mutant. Studies carried out to investigate the mechanism of virus escape from palivizumab showed a correlation between antibody binding and virus neutralization. RSV with substitutions in antigenic site A that were resistant to neutralization by palivizumab did not bind to palivizumab. 

At least one of the palivizumab resistance-associated substitutions, N262D, K272E/Q, or S275F/L was identified in 8 of 126 clinical RSV (59 RSV A and 67 RSV B) isolates from subjects who failed immunoprophylaxis, resulting in a combined resistance-associated mutation frequency of 6.3%. A review of clinical findings revealed no association between antigenic A site sequence changes and RSV disease severity among children receiving palivizumab immunoprophylaxis who develop RSV lower respiratory tract disease. 

Analysis of 254 clinical RSV isolates (145 RSV A and 109 RSV B) collected from immunoprophylaxis-naïve subjects revealed palivizumab resistance-associated substitutions in 2 (1 with N262D and 1 with S275F), resulting in a resistance-associated mutation frequency of 0.79%. 

F protein sequence variations outside antigenic site A: In addition to the sequence variations in antigenic site A known to confer palivizumab resistance, F protein substitutions T100A, G139S, N165D/V406I; T326A, V450A in RSV A, and T74I, A147V, I206L, S285G, V450I, T455I in RSV B were identified in viruses isolated from failures of immunoprophylaxis. These substitutions were not identified in RSV F sequences derived from 254 clinical isolates from immunoprophylaxis-naïve subjects and thus are considered treatment-associated and non-polymorphic. Recombinant RSV B encoding the S285G substitution exhibited palivizumab sensitivity (EC50 value = 0.39 ± 0.02 mcg per mL) similar to recombinant wild-type RSV B (EC50 value = 0.17 ± 0.02 mcg per mL). 

Palivizumab susceptibility of RSV encoding common F protein sequence polymorphisms located proximal to antigenic site A was evaluated. Recombinant RSV A encoding N276S (EC50 value = 0.72 ± 0.07 mcg per mL), and recombinant RSV B with S276N (EC50 value = 0.42 ± 0.04 mcg per mL), exhibited sensitivities comparable to the corresponding recombinant wild-type RSV A (EC50 value = 0.63 ± 0.22 mcg per mL) and RSV B (EC50 value = 0.23 ± 0.07 mcg per mL). Likewise, RSV B clinical isolates containing the polymorphic variation V278A were at least as sensitive to neutralization by palivizumab (EC50 range 0.08-0.45 mcg per mL) as laboratory strains of wild-type RSV B (EC50 value = 0.54 ± 0.08 mcg per mL). No known polymorphic or non-polymorphic sequence variations outside the antigenic site A on RSV F have been demonstrated to render RSV resistant to neutralization by palivizumab. 

Interference of RSV Diagnostic Assays by Palivizumab

Interference with immunologically-based RSV diagnostic assays by palivizumab has been observed in laboratory studies. Rapid chromatographic/enzyme immunoassays (CIA/EIA), immunofluorescence assays (IFA), and direct immunofluorescence assays (DFA) using monoclonal antibodies targeting RSV F protein may be inhibited. Therefore, caution should be used in interpreting negative immunological assay results when clinical observations are consistent with RSV infection. A reverse transcriptase-polymerase chain reaction (RT-PCR) assay, which is not inhibited by palivizumab, may prove useful for laboratory confirmation of RSV infection [see Warnings and Precautions (5.3)].